Genes and polypeptides relating to prostate cancers

ABSTRACT

The present application provides novel human gene MICAL2-PV whose expression is markedly elevated in prostate cancers. Furthermore, it provides polypeptides encoded by the gene as well as polypeptides encoded by PCOTH which expression was also discovered to be elevated in prostate cancers. The genes and polypeptides encoded by the genes can be used, for example, in the diagnosis of prostate cancers, as target molecules for developing drugs against the disease, and for attenuating cell growth of prostate cancer.

The present application is related to U.S. Ser. No. 60/414,873, filedSep. 30, 2002, which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the field of biological science, morespecifically to the field of cancer research. In particular, the presentinvention relates to novel polypeptides encoded by a novel gene A5736(MICAL2-PV) and gene D4493 (PCOTH) relating to prostate cancer.Furthermore, the present invention relates to the novel gene A5736(MICAL2-PV). The genes and polypeptides of the present invention can beused, for example, in the diagnosis of prostate cancer, as targetmolecules for developing drugs against the disease, and for attenuatingcell growth of prostate cancer.

BACKGROUND ART

Prostate cancer is one of the most common cancers in male in Westerncountries (Gronberg, Lancet 361: 859-64 (2003)). Incidence of prostatecancer is steadily increasing in developed countries due to theprevalence of Western-style diet and increasing number of seniorpopulation. Early diagnosis through serum testing for prostate specificantigen (PSA) provides an opportunity for curative surgery and hassignificantly improved the prognosis of prostate cancer. However, up to30% of patients treated with radical prostatectomy relapse cancer (Hanet al., J Urol 166: 416-9 (2001)). Most relapsed or advanced cancersrespond to androgen ablation therapy because the growth of prostatecancer is androgen-dependent in the initial stages. However, most of thepatient treated by the therapy eventually progress toandrogen-independent disease, at which point they are no longerresponsive to the therapy. The most serious clinical problem of prostatecancer is that androgen-independent prostate cancer is unresponsive toany other therapies (Gronberg, Lancet 361: 859-64 (2003)). Thus, theestablishment of new therapies other than androgen ablation therapyagainst prostate cancer is an urgent issue for the management ofprostate cancer.

cDNA microarray technologies have enabled to obtain comprehensiveprofiles of gene expression in normal and malignant cells, and comparethe gene expression in malignant and corresponding normal cells (Okabeet al., Cancer Res 61:2129-37 (2001); Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene 21:4120-8 (2002); Hasegawa et al.,Cancer Res 62:7012-7 (2002)). This approach enables to disclose thecomplex nature of cancer cells, and helps to understand the mechanism ofcarcinogenesis. Identification of genes that are deregulated in tumorscan lead to more precise and accurate diagnosis of individual cancers,and to develop novel therapeutic targets (Bienz and Clevers, Cell103:311-20 (2000)). To disclose mechanisms underlying tumors from agenome-wide point of view, and discover target molecules for diagnosisand development of novel therapeutic drugs, the present inventors havebeen analyzing the expression profiles of tumor cells using a cDNAmicroarray of 23040 genes (Okabe et al., Cancer Res 61:2129-37 (2001);Kitahara et al., Cancer Res 61:3544-9 (2001); Lin et al., Oncogene21:4120-8 (2002); Hasegawa et al., Cancer Res 62:7012-7 (2002)).

Studies designed to reveal mechanisms of carcinogenesis have alreadyfacilitated identification of molecular targets for anti-tumor agents.For example, inhibitors of farnexyltransferase (FTIs) which wereoriginally developed to inhibit the growth-signaling pathway related toRas, whose activation depends on posttranslational farnesylation, hasbeen effective in treating Ras-dependent tumors in animal models (He etal., Cell 99:33545 (1999)). Clinical trials on human using a combinationor anti-cancer drugs and anti-HER2 monoclonal antibody, trastuzumab,have been conducted to antagonize the proto-oncogene receptor HER2/neu;and have been achieving improved clinical response and overall survivalof breast-cancer patients (Lin et al., Cancer Res 61:6345-9 (2001)). Atyrosine kinase inhibitor, STI-571, which selectively inactivatesbcr-abl fusion proteins, has been developed to treat chronic myelogenousleukemias wherein constitutive activation of ber-abl tyrosine kinaseplays a crucial role in the transformation of leukocytes. Agents ofthese kinds are designed to suppress oncogenic activity of specific geneproducts (Fujita et al., Cancer Res 61:7722-6 (2001)). Therefore, geneproducts commonly up-regulated in cancerous cells may serve as potentialtargets for developing novel anti-cancer agents. In fact, novel drugstargeting abnormally expressed molecules that have causative effects oncancer growth and progression have been proven to be effective tocertain types of cancers. Such drugs include Herceptin for breastcancer, Glivec (STI571) for CML and Iressa (ZD1839) for non-small celllung cancer.

Several molecules have been known to be over-expressed in prostatecancer and are identified as therapeutic targets or markers of prostatecancer (Xu et al., Cancer Res 60: 6568-72 (2000); Luo et al., Cancer Res62: 2220-6 (2002)). However, most of them are also highly expressed inother major organs. Thus, agents that target these molecules may betoxic to cancer cells but may also adversely affect normally growingcells of other organs.

It has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs)recognize epitope peptides derived from tumor-associated antigens (TAAs)presented on MHC Class I molecule, and lyse tumor cells. Since thediscovery of MAGE family as the first example of TAAs, many other TAAshave been discovered using immunological approaches (Boon, Int J Cancer54: 177-80 (1993); Boon and van der Bruggen, J Exp Med 183: 725-9(1996); van der Bruggen et al., Science 254: 1643-7 (1991); Brichard etal., J Exp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180:347-52 (1994)). Some of the discovered TAAs are now in the stage ofclinical development as targets of immunotherapy. TAAs discovered so farinclude MAGE (van der Bruggen et al., Science 254: 1643-7 (1991)), gp100(Kawakami et al., J Exp Med 180: 347-52 (1994)), SART (Shichijo et al.,J Exp Med 187: 277-88 (1998)), and NY-ESO-1 (Chen et al., Proc Natl AcadSci USA 94: 1914-8 (1997)). On the other hand, gene products which hadbeen demonstrated to be specifically over-expressed in tumor cells, havebeen shown to be recognized as targets inducing cellular immuneresponses. Such gene products include p53 (Umano et al., Brit J Cancer84: 1052-7 (2001)), HER2/neu (Tanaka et al., Brit J Cancer 84: 94-9(2001)), CEA (Nukaya et al., Int J Cancer 80: 92-7 (1999)), and so on.

In spite of significant progress in basic and clinical researchconcerning TAAs (Rosenbeg et al., Nature Med 4: 321-7 (1998); Mukhedjiet al., Proc Natl Acad Sci USA 92: 8078-82 (1995); Hu et al., Cancer Res56: 2479-83 (1996)), only limited number of candidate TAAs for thetreatment of adenocarcinomas, including colorectal cancer, areavailable. TAAs abundantly expressed in cancer cells, and at the sametime which expression is restricted to cancer cells would be promisingcandidates as immunotherapeutic targets. Further, identification of newTAAs inducing potent and specific antitumor immune responses is expectedto encourage clinical use of peptide vaccination strategy in varioustypes of cancer (Boon and can der Bruggen, J Exp Med 183: 725-9 (1996);van der Bruggen et al., Science 254: 1643-7 (1991); Brichard et al., JExp Med 178: 489-95 (1993); Kawakami et al., J Exp Med 180: 347-52(1994); Shichijo et al., J Exp Med 187: 277-88 (1998); Chen et al., ProcNatl Acad Sci USA 94: 1914-8 (1997); Harris, J Natl Cancer Inst 88:1442-5 (1996); Butterfield et al., Cancer Res 59: 3134-42 (1999);Vissers et al., Cancer Res 59: 5554-9 (1999); van der Burg et al., JImmunol 156: 3308-14 (1996); Tanaka et al., Cancer Res 57: 4465-8(1997); Fujie et al., Int J Cancer 80: 169-72 (1999); Kikuchi et al.,Int J Cancer 81: 459-66 (1999); Oiso et al., Int J Cancer 81: 387-94(1999)).

It has been repeatedly reported that peptide-stimulated peripheral bloodmononuclear cells (PBMCs) from certain healthy donors producesignificant levels of IFN-γ in response to the peptide, but rarely exertcytotoxicity against tumor cells in an HLA-A24 or -A0201 restrictedmanner in ⁵¹Cr-release assays (Kawano et al., Cancer Res 60: 3550-8(2000); Nishizaka et al., Cancer Res 60: 4830-7 (2000); Tamura et al.,Jpn J Cancer Res 92: 762-7 (2001)). However, both of HLA-A24 andHLA-A0201 are one of the popular HLA alleles in Japanese, as well asCaucasian (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al.,J Immunol 155: 4307-12 (1995); Kubo et al., J Immunol 152: 3913-24(1994); Imanishi et al., Proceeding of the eleventh InternationalHistocompatibility Workshop and Conference Oxford University Press,Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)).Thus, antigenic peptides of cancers presented by these HLAs may beespecially useful for the treatment of cancers among Japanese andCaucasian. Further, it is known that the induction of low-affinity CTLin vitro usually results from the use of peptide at a highconcentration, generating a high level of specific peptide/MHC complexeson antigen presenting cells (APCs), which will effectively activatethese CTL (Alexander-Miller et al., Proc Natl Acad Sci USA 93: 4102-7(1996)).

SUMMARY OF THE INVENTION

To disclose the mechanism of prostate cancer and identify noveldiagnostic markers and/or drug targets for the treatment of thesetumors, the present inventors analyzed the expression profiles of genesin prostate cancer using a genome-wide cDNA microarray combined withlaser microbeam microdissection. From the pharmacological point of view,suppressing oncogenic signals is easier in practice than activatingtumor-suppressive effects. Thus, the present inventors searched forgenes that are over-expressed in prostate cancer cells.

As a result, two genes, PCOTH and MICAL2-PV, specifically over-expressedin prostate cancer cells were identified. Furthermore, reduction ofPCOTH (prostate collagen triple helix) or MICAL2-PV (MICAL2 (MoleculeInteracting with CasL 2) prostate cancer-variants) expression bytransfection of small interfering RNAs (siRNAs) inhibited the growth ofprostate cancer cells.

PCOTH encodes a 100-amino acid protein comprising a collagen triplehelix repeat and its exogenous product was localized in the cellmembrane. According to a Northern blot analysis, the expression of PCOTHwas shown to be restricted to testis and prostate.

Furthermore, the expression of MICAL2-PV was also shown to be restrictedto testis. The protein encoded by MICAL2-PV comprises a domain havinghomology to calponin domain, an actin-binding domain which is present induplicate at the N-terminus of spectrin-like proteins includingdystrophin and α-actinin. Thus, the protein encoded by MICAL2-PV ispredicted to interact with actin or other microtubles. These domainscross-link actin filaments into bundles and networks. The other familymember, MICAL1, is reported to be associated with vimentin (Suzuki etal., J Biol Chem 277: 14933-41 (2002)) and rab1 (Weide et al., BiochemBiophys Res Commun 306: 79-86 (2003)) that are major components ofintermediated filaments and cytoskelton functioning as scaffold proteinsconnecting different components in the cell. MICAL2-PV protein isexpected to be involved in the construction of cytoskelton and cellmorphology in prostate cancer cells.

Many anticancer drugs are not only toxic to cancer cells but also fornormally growing cells. However, agents suppressing the expression ofMICAL2-PV or PCOTH may not adversely affect other organs due to the factthat normal expression of MICAL2-PV is restricted to testis and PCOTH isrestricted to testis and prostate, and thus may be of great importancefor treating or preventing prostate cancer.

Thus, the present invention provides isolated genes, MICAL2-PV andPCOTH, which serve as candidates of diagnostic markers for prostatecancer as well as promising potential targets for developing newstrategies for diagnosis and effective anti-cancer agents. Furthermore,the present invention provides polypeptides encoded by these genes, aswell as the production and the use of the same. More specifically, thepresent invention provides the following:

The present application provides novel human polypeptides, MICAL2-PV andPCOTH, or a functional equivalent thereof, which expressions areelevated in prostate cancer cells.

In a preferred embodiment, the MICAL2-PV polypeptide includes a putative976 amino acid protein encoded by the open reading frame of SEQ ID NO: 3or a putative 955 amino acid protein encoded by the open reading frameof SEQ IN NO: 5. The MICAL2-PV polypeptide preferably includes the aminoacid sequence set forth in SEQ ID NO: 4 or 6. The present applicationalso provides an isolated protein encoded from at least a portion of theMICAL2-PV polynucleotide sequence, or polynucleotide sequences at least15% and more preferably at least 25% complementary to the sequence setforth in SEQ ID NO: 3 or 5.

On the other hand, in a preferred embodiment, the PCOTH polypeptideconsists of a putative 100 amino acid sequence set forth in SEQ ID NO: 2(GenBank® Accession No. AB 113650). PCOTH is encoded by the open readingframe of SEQ ID NO: 1 and comprises a collagen triple helix repeat (FIG.1(C)). The present application also provides an isolated protein encodedfrom at least a portion of the PCOTH polynucleotide sequence, orpolynucleotide sequences at least 30% and more preferably at least 40%complementary to the sequence set forth in SEQ ID NO: 1(LOC221179(XP_(—)167955)).

The present invention further provides a novel human gene MICAL2-PVwhose expressions is markedly elevated in a great majority of prostatecancers as compared to corresponding non-cancerous prostate ductepithelium. The isolated MICAL2-PV gene includes a polynucleotidesequence as described in SEQ ID NO: 3 or 5. In particular, the MICAL2-PVcDNA includes 6805 nucleotides that contain an open reading frame of2928 nucleotides (SEQ ID NO: 3) or 6742 nucleotides that contain an openreading frame of 2865 nucleotides (SEQ ID NO: 5). The present inventionfurther encompasses polynucleotides which hybridize to and which are atleast 30% and more preferably at least 40% complementary to thepolynucleotide sequence set forth in SEQ ID NO: 3 or 5, to the extentthat they encode a MICAL2-PV protein or a functional equivalent thereof.Examples of such polynucleotides are degenerates and allelic mutants ofMICAL2-PV encoded by the sequence of SEQ ID NO: 3 or 5.

Furthermore, the present invention provides an isolated polynucleotideencoding the novel human protein PCOTH, whose expression is alsomarkedly elevated in a great majority of prostate cancers as compared tocorresponding non-cancerous prostate duct epithelium. The isolatedpolynucleotide encodes a polypeptide consisting of 100 amino acidsdescribed in SEQ ID NO: 2. More specifically, the isolatedpolynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 fromthe 332 to the 631 nucleotide. The present invention further encompassespolynucleotides which hybridize to and which are at least 30%, and morepreferably at least 40% complementary to the polynucleotide sequence setforth in SEQ ID NO: 1, to the extent that they encode a PCTOH protein ora functional equivalent thereof. Examples of such polynucleotides aredegenerates and allelic mutants of SEQ ID NO: 1.

As used herein, an isolated gene is a polynucleotide the structure ofwhich is not identical to that of any naturally occurring polynucleotideor to that of any fragment of a naturally occurring genomicpolynucleotide spanning more than three separate genes. The termtherefore includes, for example, (a) a DNA which has the sequence ofpart of a naturally occurring genomic DNA molecule in the genome of theorganism in which it naturally occurs; (b) a polynucleotide incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion polypeptide.

Accordingly, in one aspect, the invention provides an isolatedpolynucleotide that encodes a polypeptide described herein or a fragmentthereof. Preferably, the isolated polypeptide includes a nucleotidesequence that is at least 60% identical to the nucleotide sequence shownin SEQ ID NO: 1, 3 or 5. More preferably, the isolated nucleic acidmolecule is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%,98%, 99% or more, identical to the nucleotide sequenceshown in SEQ ID NO: 1, 3 or 5. In the case of an isolated polynucleotidewhich is longer than or equivalent in length to the reference sequence,e.g., SEQ ID NO: 1, 3 or 5, the comparison is made with the full lengthof the reference sequence. Where the isolated polynucleotide is shorterthan the reference sequence, e.g., shorter than SEQ ID NO: 1, 3 or 5,the comparison is made to segment of the reference sequence of the samelength (excluding any loop required by the homology calculation).

The present invention also provides a method of producing a protein bytransfecting or transforming a host cell with a polynucleotide sequenceencoding the MICAL2-PV or PCOTH protein, and expressing thepolynucleotide sequence. In addition, the present invention providesvectors comprising a nucleotide sequence encoding the MICAL2-PV or PCOTHprotein, and host cells harboring a polynucleotide encoding theMICAL2-PV or PCOTH protein. Such vectors and host cells may be used forproducing the MICAL2-PV or PCOTH protein.

An antibody that recognizes the MICAL2-PV protein is also provided bythe present application. In part, an antisense polynucleotide (e.g.,antisense DNA), ribozyme, and siRNA (small interfering RNA) of theMICAL2-PV or PCOTH gene is also provided.

The present invention further provides a method for diagnosis ofprostate cancer which includes the step of determining an expressionlevel of the gene in a biological sample from a subject, comparing theexpression level of MICAL2-PV or PCOTH gene with that in a normalsample, and defining that a high expression level of the MICAL2-PV orPCOTH gene in the sample indicates that the subject suffers from or isat risk of developing prostate cancer.

Further, a method of screening for a compound for treating or preventingprostate cancer is provided by the present invention. The methodincludes contacting the MICAL2-PV or PCOTH polypeptide with testcompounds, and selecting test compounds that bind to or that alter thebiological activity of the MICAL2-PV or PCOTH polypeptide.

The present invention further provides a method of screening for acompound for treating or preventing prostate cancer, wherein the methodincludes contacting a test compound with a cell expressing the MICAL2-PVor PCOTH polypeptide or introduced with a vector comprising thetranscriptional regulatory region of MICAL2-PV or PCOTH upstream of areporter gene, and selecting the test compound that suppresses theexpression level of the MICAL2-PV or PCOTH polypeptide.

Alternatively, the present invention provides a method of screening fora compound for treating or preventing prostate cancer, wherein themethod includes contacting MICAL2-PV and actin in the presence of a testcompound, and selecting the test compound that inhibits the binding ofMICAL2-PV and actin.

The present application also provides a pharmaceutical composition fortreating or preventing prostate cancer. The pharmaceutical compositionmay be, for example, an anti-cancer agent. The pharmaceuticalcomposition can be described as at least a portion of the antisenseS-oligonucleotides, siRNA or ribozyme against the MICAL2-PV or PCOTHpolynucleotide sequence shown and described in SEQ ID NOs: 3 and 5, or1, respectively. A suitable siRNA targets a sequence selected from thegroup of SEQ ID NOs: 23 and 27. The target sequence of MICAL2-PV siRNAcomprises the nucleotide sequence of SEQ ID NO: 27, and that of PCOTHsiRNA comprises the nucleotide sequence of SEQ ID NO: 23. Both may bepreferably selected as targets for treating or preventing prostatecancer according to the present invention. The pharmaceuticalcompositions may be also those comprising the compounds selected by thepresent methods of screening for compounds for treating or preventingcell proliferative diseases such as prostate cancer.

The course of action of the pharmaceutical composition is desirably toinhibit growth of the cancerous cells such as prostate cancer cells. Thepharmaceutical composition may be applied to mammals including humansand domesticated mammals.

The present invention further provides methods for treating orpreventing prostate cancer using the pharmaceutical composition providedby the present invention.

In addition, the present invention provides method for treating orpreventing cancer, which method comprises the step of administering theMICAL2-PV or PCOTH polypeptide. It is expected that anti tumor immunitybe induced by the administration of the MICAL2-PV or PCOTH polypeptide.Thus, the present invention also provides method for inducing anti tumorimmunity, which method comprises the step of administering the MICAL2-PVor PCOTH polypeptide, as well as pharmaceutical composition for treatingor preventing cancer comprising the MICAL2-PV or PCOTH polypeptide.

It is to be understood that both the foregoing summary of the inventionand the following detailed description are of a preferred embodiment,and not restrictive of the invention or other alternate embodiments ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) depicts photographs showing the result of validation ofover-expression of D4493 (PCOTH) in prostate cancer cells by RT-PCR. Themicrodissected normal prostate duct epithelial cells (N) and prostatecancer cells (T) from the same individual were compared bysemiquantitative RT-PCR. ACTB was used for normalization of the results.(B) depicts photographs showing the result of Northern blot analysis ofnormal human multiple tissues. High and localized expression in testisand prostate and minor expression in heart and bone marrow weredetected. (C) depicts the amino acid sequence of D4493 (PCOTH) product(SEQ ID NO:2). The product consists of 100 amino acids and has collagentriple helix repeats which is characterized by the G-X-X motif repeat. Gis glycine and X is preferably proline.

FIG. 2(A) depicts photographs showing the result of validation ofover-expression of A5736 (MICAL2-PV) in prostate cancer cells by RT-PCR.The microdissected normal prostate duct epithelial cells (N) andprostate cancer cells (T) from the same individual were compared bysemiquantitative RT-PCR. ACTB was used for normalization of the results.(B) depicts photographs showing the result of Northern blot analysis ofnormal human multiple tissues and prostate cancer cell lines. Theapproximately 7 kb transcript corresponding to MICAL2-PV was highlyexpressed in testis and prostate cancer cell lines (LNCaP, PC3 andDU145), while the approximately 4 kb transcript corresponding to theoriginal MICAL2 was expressed in heart, brain and liver, but not inprostate cancer cell lines. (C) depicts an illustration showing thealignment of the exons of MICAL2 (KIAA0750) and MICAL2-PV. MICAL2(KIAA0750) is a 3.8 kb transcript and consists of 28 exons, whileMICAL2-PV is a 6.8 kb transcript in which several exons are deleted andconsist of the long last exons. MICAL2-PV has two isoforms, long form(Accession number: AB110785) and short form (Accession number: AB110786)wherein one exon is deleted from the long form. The long form ispredicted to yield a 976-amino acid protein that is different from theMICAL2 (KIAA0750) protein in its COOH region.

FIG. 3 depicts photographs showing the sublocalization of exogenousPCOTH protein (A) and MICAL2-PV protein (B) in COS7 cells. ExogenousPCOTH protein was localized in the cell membrane, while exogenousMICAL2-PV protein was localized in the cytoplasm of COS7 cells.

FIG. 4(A) depicts photographs showing the effect of knocking-down PCOTHin prostate cancer cell line using siRNA. Several U6-promoter-siRNAconstructs (sil-4 targeting PCOTH and one targeting EGFP) weretransfected to PC3. The result of RT-PCR demonstrated that a drasticeffect is achieved by knocking-down PCOTH in PC3 cells transfected withsi3. (B) depicts a photograph showing the result of colony formationassay in PC3 after the transfection with U6-promoter-siRNA constructs.The number of colonies was concordant with the knocking-down effect ofsi3 on PCOTH. (C) depicts the result of MTT assay in three prostatecancer cell lines (LNCaP, DU145 and PC3) after knocking-down PCOTH withsiRNA. The cell growth of cells was also concordant with theknocking-down effect of si3 on PCOTH. (D) depicts photographs showingthe effect of knocking-down MICAL2-PV in prostate cancer cell line withsiRNA. Several U6-promoter-siRNA constructs (sil-3 targeting MICAL2-PVand one targeting EGFP) were transfected into PC3. The result of RT-PCRshowed a drastic effect of knocking-down PCOTH in PC3 cells by thetransfection with si2. (E) depicts photographs showing the result ofcolony formation assay on PC3 after the transfection withU6-promoter-siRNA constructs. The number of colonies was concordant withthe knocking-down effect of si2 on MICAL2-PV.

DETAILED DESCRIPTION OF THE INVENTION

The words “a”, “an” and “the” as used herein mean “at least one” unlessotherwise specifically indicated.

To disclose the mechanism of prostate cancer and identify noveldiagnostic markers and/or drug targets for the treatment of thesetumors, the present inventors analyzed the expression profiles of genesin prostate cancer using a genome-wide cDNA microarray combined withlaser microbeam microdissection. As a result, two genes, PCOTH andMICAL2-PV, specifically over-expressed in prostate cancer cells wereidentified. Furthermore, suppression of the expression of PCOTH orMICAL2-PV gene with small interfering RNAs (siRNAs) resulted in asignificant growth-inhibition of cancerous cells. These findings suggestthat PCOTH and MICAL2-PV render oncogenic activities to cancer cells,and that inhibition of the activity of these proteins could be apromising strategy for the treatment and prevention of proliferativediseases such as prostate cancers.

MICAL2-PV

According to the present invention, two genes with a similar sequencewere identified and suggested to encode variants of MICAL2 that consistof 1124 amino acid residues. The expression of the two genes wasmarkedly elevated in prostate cancer compared to correspondingnon-cancerous tissues. The coding region of the 3′ terminus of theidentified variants differed from that of MICAL2. Thus, these two novelhuman genes were collectively dubbed “MICAL2 prostate cancer-variants(MICAL2-PV)”. The cDNA of the longer variant consists of 6805nucleotides containing an open reading frame of 2928 nucleotides (SEQ IDNO: 3) and the shorter variant consists of 6742 nucleotides containingan open reading frame of 2865 nucleotides (SEQ ID NO: 5). These openreading frames encode a putative 976 amino acid-protein and a putative955 amino acid-protein, respectively. The protein encoded by MICAL2-PVcomprises a domain having homology to calponin domain, an actin-bindingdomain which is present in duplicate at the N-terminus of spectrin-likeproteins including dystrophin and α-actinin. Therefore, the proteinencoded by MICAL2-PV is predicted to interact with actin or othermicrotubles. These domains cross-link actin filaments into bundles andnetworks.

Thus, the present invention provides substantially pure polypeptidesencoded by these genes including polypeptides comprising the amino acidsequence of SEQ ID NO: 4 or 6, as well as functional equivalentsthereof, to the extent that they encode a MICAL2-PV protein. Examples ofpolypeptides functionally equivalent to MICAL2-PV include, for example,homologous proteins of other organisms corresponding to the humanMICAL2-PV protein, as well as mutants of human MICAL2-PV proteins.

In the present invention, the term “functionally equivalent” means thatthe subject polypeptide has the activity to promote cell proliferationlike the MICAL2-PV protein and to confer oncogenic activity to cancercells. Whether the subject polypeptide has a cell proliferation activityor not can be judged by introducing the DNA encoding the subjectpolypeptide into a cell expressing the respective polypeptide anddetecting promotion of proliferation of the cells or increase in colonyforming activity. Such cells include, for example, LNCaP, PC3 and DU145.Alternatively, whether the subject polypeptide is functionallyequivalent to MICAL2-PV may be judged by detecting its binding abilityto actin.

Methods for preparing polypeptides functionally equivalent to a givenprotein are well known by a person skilled in the art and include knownmethods of introducing mutations into the protein. For example, oneskilled in the art can prepare polypeptides functionally equivalent tothe human MICAL2-PV protein by introducing an appropriate mutation inthe amino acid sequence of either of these proteins by site-directedmutagenesis (Hashimoto-Gotoh et al., Gene 152:271-5 (1995); Zoller andSmith, Methods Enzymol 100: 468-500 (1983); Kramer et al., Nucleic AcidsRes. 12:9441-9456 (1984); Kramer and Fritz, Methods Enzymol 154: 350-67(1987); Kunkel, Proc Natl Acad Sci USA 82: 488-92 (1985); Kunkel,Methods Enzymol 85: 2763-6 (1988)). Amino acid mutations can occur innature, too. The polypeptide of the present invention includes thoseproteins having the amino acid sequences of the human MICAL2-PV proteinin which one or more amino acids are mutated, provided the resultingmutated polypeptides are functionally equivalent to the human MICAL2-PVprotein. The number of amino acids to be mutated in such a mutant isgenerally 10 amino acids or less, preferably 6 amino acids or less, andmore preferably 3 amino acids or less.

Mutated or modified proteins, proteins having amino acid sequencesmodified by substituting, deleting, inserting and/or adding one or moreamino acid residues of a certain amino acid sequence, have been known toretain the original biological activity (Mark et al., Proc Natl Acad SciUSA 81: 5662-6 (1984); Zoller and Smith, Nucleic Acids Res 10:6487-500(1982); Dalbadie-McFarland et al., Proc Natl Acad Sci USA 79: 6409-13(1982)).

The amino acid residue to be mutated is preferably mutated into adifferent amino acid in which the properties of the amino acidside-chain are conserved (a process known as conservative amino acidsubstitution). Examples of properties of amino acid side chains arehydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic aminoacids (R, D, N, C, E, Q, C H, K, S, T), and side chains having thefollowing functional groups or characteristics in common: an aliphaticside-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain(S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acidand amide containing side-chain (D, N, E, Q); a base containingside-chain (R, K, H); and an aromatic containing side-chain (H, F, Y,W). Note, the parenthetic letters indicate the one-letter codes of aminoacids.

An example of a polypeptide to which one or more amino acids residuesare added to the amino acid sequence of human MICAL2-PV protein is afusion protein containing the human MICAL2-PV protein. Fusion proteinsare, fusions of the human MICAL2-PV protein and other peptides orproteins, and are included in the present invention. Fusion proteins canbe made by techniques well known to a person skilled in the art, such asby linking the DNA encoding the human MICAL2-PV protein of the inventionwith DNA encoding other peptides or proteins, so that the frames match,inserting the fusion DNA into an expression vector and expressing it ina host. There is no restriction as to the peptides or proteins fused tothe protein of the present invention.

Known peptides that can be used as peptides that are fused to theprotein of the present invention include, for example, FLAG (Hopp etal., Biotechnology 6: 1204-10 (1988)), 6×His containing six His(histidine) residues, 10×His, Influenza agglutinin (HA), human c-mycfragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag,SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment and the like. Examples of proteins that may be fused to aprotein of the invention include GST (glutathione-S-transferase),Influenza agglutinin (HA), immunoglobulin constant region,β-galactosidase, MBP (maltose-binding protein) and such.

Fusion proteins can be prepared by fusing commercially available DNA,encoding the fusion peptides or proteins discussed above, with the DNAencoding the polypeptide of the present invention and expressing thefused DNA prepared.

An alternative method known in the art to isolate functionallyequivalent polypeptides is, for example, the method using ahybridization technique (Sambrook et al., Molecular Cloning 2nd ed.9.47-9.58, Cold Spring Harbor Lab. Press (1989)). One skilled in the artcan readily isolate a DNA having high homology with a whole or part ofthe DNA sequence encoding the human MICAL2-PV protein (i.e., SEQ ID NO:3 or 5), and isolate functionally equivalent polypeptides to the humanMICAL2-PV protein from the isolated DNA. The polypeptides of the presentinvention include those that are encoded by DNA that hybridize with awhole or part of the DNA sequence encoding the human MICAL2-PV proteinand are functionally equivalent to the human MICAL2-PV protein. Thesepolypeptides include mammal homologues corresponding to the proteinderived from human (for example, a polypeptide encoded by a monkey, rat,rabbit and bovine gene). In isolating a cDNA highly homologous to theDNA encoding the human MICAL2-PV protein from animals, it isparticularly preferable to use tissues from testis.

The condition of hybridization for isolating a DNA encoding apolypeptide functionally equivalent to the human MICAL2-PV protein canbe routinely selected by a person skilled in the art. For example,hybridization may be performed by conducting prehybridization at 68° C.for 30 min or longer using “Rapid-hyb buffer” (Amersham LIFE SCIENCE),adding a labeled probe, and warming at 68° C. for 1 hour or longer. Thefollowing washing step can be conducted, for example, in a low stringentcondition. A low stringent condition is, for example, 42° C., 2×SSC,0.1% SDS, or preferably 50° C., 2×SSC, 0.1% SDS. More preferably, highstringent conditions are used. A high stringent condition is, forexample, washing 3 times in 2×SSC, 0.01% SDS at room temperature for 20min, then washing 3 times in 1×SSC, 0.1% SDS at 37° C. for 20 min, andwashing twice in 1×SSC, 0.1% SDS at 50° C. for 20 min. However, severalfactors, such as temperature and salt concentration, can influence thestringency of hybridization and one skilled in the art can suitablyselect the factors to achieve the requisite stringency.

In place of hybridization, a gene amplification method, for example, thepolymerase chain reaction (PCR) method, can be utilized to isolate a DNAencoding a polypeptide functionally equivalent to the human MICAL2-PVprotein, using a primer synthesized based on the sequence information ofthe protein encoding DNA (SEQ ID NO: 3 or 5).

Polypeptides that are functionally equivalent to the human MICAL2-PVprotein encoded by the DNA isolated through the above hybridizationtechniques or gene so amplification techniques normally have a highhomology to the amino acid sequence of the human MICAL2-PV protein.“High homology” typically refers to a homology of 40% or higher,preferably 60% or higher, more preferably 80% or higher, even morepreferably 95% or higher. The homology of a polypeptide can bedetermined by following the algorithm in “Wilbur and Lipman, Proc NatlAcad Sci USA 80: 726-30 (1983)”. A polypeptide of the present inventionmay have variations in amino acid sequence, molecular weight,isoelectric point, the presence or absence of sugar chains, or form,depending on the cell or host used to produce it or the purificationmethod utilized. Nevertheless, so long as it has a function equivalentto that of the human MICAL2-PV protein of the present invention, it iswithin the scope of the present invention.

The polypeptides of the present invention can be prepared as recombinantproteins or natural proteins, by methods well known to those skilled inthe art. A recombinant protein can be prepared by inserting a DNA, whichencodes the polypeptide of the present invention (for example, the DNAcomprising the nucleotide sequence of SEQ ID NO: 3 or 5), into anappropriate expression vector, introducing the vector into anappropriate host cell, obtaining the extract, and purifying thepolypeptide by subjecting the extract to chromatography, e.g., ionexchange chromatography, reverse phase chromatography, gel filtration oraffinity chromatography utilizing a column to which antibodies againstthe protein of the present invention is fixed or by combining more thanone of aforementioned columns.

Also when the polypeptide of the present invention is expressed withinhost cells (for example, animal cells and E. coli) as a fusion proteinwith glutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column.Alternatively, when the polypeptide of the present invention isexpressed as a protein tagged with c-myc, multiple histidines or FLAG,it can be detected and purified using antibodies to c-myc, His or FLAGrespectively.

After purifying the fusion protein, it is also possible to excluderegions other than the objective polypeptide by cutting with thrombin orfactor-Xa as required.

A natural protein can be isolated by methods known to a person skilledin the art, for example, by contacting the affinity column, in whichantibodies binding to the MICAL2-PV protein described below are bound,with the extract of tissues or cells expressing the polypeptide of thepresent invention. The antibodies can be polyclonal antibodies ormonoclonal antibodies.

The present invention also encompasses partial peptides of thepolypeptide of the present invention. The partial peptide has an aminoacid sequence specific to the polypeptide of the present invention andconsists of at least 7 amino acids, preferably 8 amino acids or more,and more preferably 9 amino acids or more. The partial peptide can beused, for example, for preparing antibodies against the polypeptide ofthe present invention, screening for a compound that binds to thepolypeptide of the present invention, and screening for accelerators orinhibitors of the polypeptide of the present invention.

A partial peptide of the invention can be produced by geneticengineering, by known methods of peptide synthesis or by digesting thepolypeptide of the invention with an appropriate peptidase. For peptidesynthesis, for example, solid phase synthesis or liquid phase synthesismay be used.

The present invention further provides polynucleotides that encode suchMICAL2-PV polypeptides described above. The polynucleotides of thepresent invention can be used for the in vivo or in vitro production ofthe polypeptide of the present invention as described above, or can beapplied to gene therapy for diseases attributed to genetic abnormalityin the gene encoding the protein of the present invention. Any form ofthe polynucleotide of the present invention can be used so long as itencodes the polypeptide of the present invention, including mRNA, RNA,cDNA, genomic DNA, chemically synthesized polynucleotides. Thepolynucleotide of the present invention include a DNA comprising a givennucleotide sequences as well as its degenerate sequences, so long as theresulting DNA encodes a polypeptide of the present invention.

The polynucleotide of the present invention can be prepared by methodsknown to a person skilled in the art. For example, the polynucleotide ofthe present invention can be prepared by: preparing a cDNA library fromcells which express the polypeptide of the present invention, andconducting hybridization using a partial sequence of the DNA of thepresent invention (for example, SEQ ID NO: 3 or 5) as a probe. A cDNAlibrary can be prepared, for example, by the method described inSambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press(1989); alternatively, commercially available cDNA libraries may beused. A cDNA library can be also prepared by: extracting RNAs from cellsexpressing the polypeptide of the present invention, synthesizing oligoDNAs based on the sequence of the DNA of the present invention (forexample, SEQ ID NO: 3 or 5), conducting PCR using the oligo DNAs asprimers, and amplifying cDNAs encoding the protein of the presentinvention.

In addition, by sequencing the nucleotides of the obtained cDNA, thetranslation region encoded by the cDNA can be routinely determined, andthe amino acid sequence of the polypeptide of the present invention canbe easily obtained. Moreover, by screening the genomic DNA library usingthe obtained cDNA or parts thereof as a probe, the genomic DNA can beisolated.

More specifically, mRNAs may first be prepared from a cell, tissue ororgan (e.g., testis) in which the object polypeptide of the invention isexpressed. Known methods can be used to isolate mRNAs; for instance,total RNA may be prepared by guanidine ultracentrifugation (Chirgwin etal., Biochemistry 18:5294-9 (1979)) or AGPC method (Chomczynski andSacchi, Anal Biochem 162:156-9 (1987)). In addition, mRNA may bepurified from total RNA using mRNA Purification Kit (Pharmacia) andsuch. Alternatively, mRNA may be directly purified by QuickPrep mRNAPurification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized using a commercially availablekit, such as the AMV Reverse Transcriptase First-strand cDNA SynthesisKit (Seikagaku Kogyo). Alternatively, cDNA may be synthesized andamplified following the 5′-RACE method (Frohman et al., Proc Natl AcadSci USA 85: 8998-9002 (1988); Belyavsky et al., Nucleic Acids Res 17:2919-32 (1989)), which uses a primer and such, described herein, the5′-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction(PCR).

A desired DNA fragment is prepared from the PCR products and ligatedwith a vector DNA, The recombinant vectors are used to transform E. coliand such, and a desired recombinant vector is prepared from a selectedcolony. The nucleotide sequence of the desired DNA can be verified byconventional methods, such as dideoxynucleotide chain termination.

The nucleotide sequence of a polynucleotide of the invention may bedesigned to be expressed more efficiently by taking into account thefrequency of codon usage in the host to be used for expression (Granthamet al., Nucleic Acids Res 9: 43-74 (1981)). The sequence of thepolynucleotide of the present invention may be altered by a commerciallyavailable kit or a conventional method. For instance, the sequence maybe altered by digestion with restriction enzymes, insertion of asynthetic oligonucleotide or an appropriate polynucleotide fragment,addition of a linker, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA or TAG).

Specifically, the polynucleotide of the present invention encompassesthe DNA comprising the nucleotide sequence of SEQ ID NO: 3 or 5.

Furthermore, the present invention provides a polynucleotide thathybridizes under stringent conditions with a polynucleotide having anucleotide sequence of SEQ ID NO: 3 or 5, and encodes a polypeptidefunctionally equivalent to the MICAL2-PV protein of the inventiondescribed above. One skilled in the art may appropriately choosestringent conditions. For example, low stringent condition can be used.More preferably, high stringent condition can be used. These conditionsare the same as that described above. The hybridizing DNA above ispreferably a cDNA or a chromosomal DNA.

The present invention also provides a polynucleotide which iscomplementary to the polynucleotide encoding human MICAL2-PV protein(SEQ ID NO: 3 or 5) or the complementary strand thereof, and whichcomprises at least 15 nucleotides. The polynucleotide of the presentinvention is preferably a polynucleotide which specifically hybridizeswith the DNA encoding the MICAL2-PV polypeptide of the presentinvention. The term “specifically hybridize” as used herein, means thatcross-hybridization does not occur significantly with DNA encoding otherproteins, under the usual hybridizing conditions, preferably understringent hybridizing conditions. Such polynucleotides include, probes,primers, nucleotides and nucleotide derivatives (for example, antisenseoligonucleotides and ribozymes), which specifically hybridize with DNAencoding the polypeptide of the invention or its complementary strand.Moreover, such polynucleotide can be utilized for the preparation of DNAchip.

PCOTH

According to the present invention another gene, PCOTH, was alsoidentified to be specifically over-expressed in prostate cancer cellscompared to corresponding non-cancerous tissues. The identified gene wasidentical with LOC221179 (XP₁₃ 167955). However, the PCOTH gene wasrevealed to encode a 100-amino acid protein set forth in SEQ ID NO: 2(GenBank® Accession No. AB113650) encoded by the open reading frameconsisting of 300 nucleotides shown in SEQ ID NO: 1 which differed fromthat known for LOC221179 (XP_(—)167955). PCOTH was shown to comprise acollagen triple helix repeat and its exogenous product was localized inthe cell membrane (FIG. 1). Therefore, the gene was dubbed “prostatecollagen triple helix”.

Thus, the present invention provides substantially pure polypeptidesencoded by the gene including polypeptides consisting of the amino acidsequence of SEQ ID NO: 2, as well as functional equivalents thereof, tothe extent that they encode a PCOTH protein and such functionalequivalents are expected to be shorter than the whole amino acidsequence encoded by the known LOC221179 (XP_(—)167955). Examples ofpolypeptides functionally equivalent to PCOTH include, for example,homologous proteins of other organisms corresponding to the human PCOTHprotein, as well as mutants of human PCOTH proteins. Preferable mutantsof PCOTH protein includes those consisting of the amino acid sequence ofSEQ ID NO: 2 in which one or more amino acids are substituted and/ordeleted.

In the present invention, the term “functionally equivalent” means thatthe subject polypeptide has the activity to promote cell proliferationlike the PCOTH protein and to confer oncogenic activity to cancer cells.Whether the subject polypeptide has a cell proliferation activity or notcan be judged by introducing the DNA encoding the subject polypeptideinto a cell expressing the respective polypeptide and detectingpromotion of proliferation of the cells or increase in colony formingactivity. Such cells include, for example, LNCaP, PC3 and DU145.

The same methods as those described in the item of “MICAL2-PV” above canbe employed for preparing the PCOTH protein and functional equivalentsthereof using sequence information described in SEQ ID NOs: 1 and 2.

The present invention further provides polynucleotides that encode suchPCOTH polypeptides described above. The polynucleotides of the presentinvention can be used for the in vivo or in vitro production of thepolypeptide of the present invention as described above, or can beapplied to gene therapy for diseases attributed to genetic abnormalityin the gene encoding the protein of the present invention. Any form ofthe polynucleotide of the present invention can be used so long as itencodes the polypeptide of the present invention, including mRNA, RNA,cDNA, genomic DNA, chemically synthesized polynucleotides. Thepolynucleotide of the present invention include a DNA comprising a givennucleotide sequences as well as its degenerate sequences, so long as theresulting DNA encodes a PCOTH polypeptide of the present invention. Suchpolynucleotides can also be prepared according to method similar tothose described under the item of “MICAL2-PV” using sequence informationdescribed in SEQ ID NOs: 1 and 2.

In contrast to MICAL2-PV, normal expression of PCOTH was detected intestis and also prostate. Thus, for preparing PCOTH or functionallyequivalents thereof, or the mRNA of PCOTH, in addition to testistissues, one can use tissues from prostate.

Vectors and Host Cells

The present invention also provides a vector and host cell into which apolynucleotide of the present invention is introduced. A vector of thepresent invention is useful to keep a polynucleotide, especially a DNA,of the present invention in host cell, to express the polypeptide of thepresent invention, or to administer the polynucleotide of the presentinvention for gene therapy.

When E. coli is a host cell and the vector is amplified and produced ina large amount in E. coli (e.g., JM109, DH5α, HB101 or XL1Blue), thevector should have “ori” to be amplified in E. coli and a marker genefor selecting transformed E. coli (e.g., a drug-resistance gene selectedby a: drug such as ampicillin, tetracycline, kanamycin, chloramphenieolor the like). For example, M13-series vectors, pUC-series vectors,pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T,pDIRECT and pT7 can also be used for subcloning and extracting eDNA aswell as the vectors described above. When a vector is used to producethe protein of the present invention, an expression vector is especiallyuseful. For example, an expression vector to be expressed in E. colishould have the above characteristics to be amplified in E. coli. WhenE. coli, such as JM109, DH5α, HB101 or XL1 Blue, are used as a hostcell, the vector should have a promoter, for example, lacZ promoter(Ward et al., Nature 341:544-6 (1989); FASEB J 6:2422-7 (1992)), araBpromoter (Better et al., Science 240:1041-3 (1988)), T7 promoter or thelike, that can efficiently express the desired gene in E. coli. In thatrespect, pGEX-5X-1 (Pharmacia), “QIAexpress™ system” (Qiagen), pEGFP andpET (in this case, the host is preferably BL21 which expresses T7 RNApolymerase), for example, can be used instead of the above vectors.Additionally, the vector may also contain a signal sequence forpolypeptide secretion. An exemplary signal sequence that directs thepolypeptide to be secreted to the periplasm of the E. coli is the pelBsignal sequence (Lei et al., J Bacteriol 169:4379 (1987)). Means forintroducing of the vectors into the target host cells include, forexample, the calcium chloride method, and the electroporation method.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic AcidsRes 18(17): 5322 (1990)), pEF, pCDM8), expression vectors derived frominsect cells (for example, “Bac-to-BAC baculovirus expression system”(GIBCO BRL), pBacPAK8), expression vectors derived from plants (e.g.,pMH1, pMH2), expression vectors derived from animal viruses (e.g., pHSV,pMV, pAdexLcw), expression vectors derived from retroviruses (e.g.,pZIpneo), expression vector derived from yeast (e.g., “Pichia ExpressionKit” (Invitrogen), pNV11, SP-Q01) and expression vectors derived fromBacillus subtilis (e.g., pPL608, pKTH50) can be used for producing thepolypeptide of the present invention.

In order to express the vector in animal cells, such as CHO, COS orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al., Nature277:,108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima etal., Nucleic Acids Res 18: 5322 (1990)), the CMV promoter and the like,and preferably a marker gene for selecting transformants (for example, adrug resistance gene selected by a drug (e.g., neomycin, G418)).Examples of known vectors with these characteristics include, forexample, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13.

Producing Polypeptides

In addition, the present invention provides methods for producing apolypeptide of the present invention. The polypeptides may be preparedby culturing a host cell which harbors a expression vector comprising agene encoding the polypeptide. According to needs, methods may be usedto express a gene stably and, at the same time, to amplify the copynumber of the gene in cells. For example, a vector comprising thecomplementary DHFR gene (e.g., pCHO I) may be introduced into CHO cellsin which the nucleic acid synthesizing pathway is deleted, and thenamplified by methotrexate (MTX). Furthermore, in case of transientexpression of a gene, the method wherein a vector comprising areplication origin of SV40 (pcD, etc.) is transformed into COS cellscomprising the SV40 T antigen expressing gene on the chromosome can beused.

A polypeptide of the present invention obtained as above may be isolatedfrom inside or outside (such as medium) of host cells and purified as asubstantially pure homogeneous polypeptide. The term “substantiallypure” as used herein in reference to a given polypeptide means that thepolypeptide is substantially free from other biological macromolecules.The substantially pure polypeptide is at least 75% (e.g., at least 80,85, 95, or 99%) pure by dry weight. Purity can be measured by anyappropriate standard method, for example by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis. The method forpolypeptide isolation and purification is not limited to any specificmethod; in fact, any standard method may be used.

For instance, column chromatography, filter, ultrafiltration, saltprecipitation, solvent precipitation, solvent extraction, distillation,immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectricpoint electrophoresis, dialysis, and recrystallization may beappropriately selected and combined to isolate and purify thepolypeptide.

Examples of chromatography include, for example, affinitychromatography, ion-exchange chromatography, hydrophobic chromatography,gel filtration, reverse phase chromatography, adsorption chromatography,and such (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed. Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). These chromatographies may be performedby liquid chromatography, such as HPLC and FPLC. Thus, the presentinvention provides for highly purified-polypeptides prepared by theabove methods.

A polypeptide of the present invention may be optionally modified orpartially deleted by treating it with an appropriate proteinmodification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, glucosidase and so on.

Antibodies

The present invention provides an antibody that binds to the polypeptideof the invention. The antibody of the invention can be used in any form,such as monoclonal or polyclonal antibodies, and includes antiserumobtained by immunizing an animal such as a rabbit with the polypeptideof the invention, all classes of polyclonal and monoclonal antibodies,human antibodies and humanized antibodies produced by geneticrecombination.

A polypeptide of the invention used as an antigen to obtain an antibodymay be derived from any animal species, but preferably is derived from amammal such as a human, mouse, or rat, more preferably from a human. Ahuman-derived polypeptide may be obtained from the nucleotide or aminoacid sequences disclosed herein. According to the present invention, thepolypeptide to be used as an immunization antigen may be a completeprotein or a partial peptide of the protein. A partial peptide maycomprise, for example, the amino (N)-terminal or carboxy (C)-terminalfragment of a polypeptide of the present invention.

Herein, an antibody is defined as a protein that reacts with either thefull length or a fragment of a polypeptide of the present invention.

A gene encoding a polypeptide of the invention or its fragment may beinserted into a known expression vector, which is then used to transforma host cell as described herein. The desired polypeptide or its fragmentmay be recovered from the outside or inside of host cells by anystandard method, and may subsequently be used as an antigen.Alternatively, whole cells expressing the polypeptide or their lysatesor a chemically synthesized polypeptide may be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha or Primates areused. Animals of Rodentia include, for example, mouse, rat and hamster.Animals of Lagomorpha include, for example, rabbit Animals of Primatesinclude, for example, a monkey of Catarrhini (old world monkey) such asMacaca fascicularis, rhesus monkey, sacred baboon and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for immunization of mammals. More specifically, antigensmay be diluted and suspended in an appropriate amount of phosphatebuffered saline (PBS), physiological saline, etc. If desired, theantigen suspension may be mixed with an appropriate amount of a standardadjuvant, such as Freund's complete adjuvant, made into emulsion andthen administered to mammalian animals. Preferably, it is followed byseveral administrations of antigen mixed with an appropriately amount ofFreund's incomplete adjuvant every 4 to 21 days. An appropriate carriermay also be used for immunization. After immunization as above, serum isexamined by a standard method for an increase in the amount of desiredantibodies.

Polyclonal antibodies against the polypeptides of the present inventionmay be prepared by collecting blood from the immunized mammal examinedfor the increase of desired antibodies in the serum, and by separatingserum from the blood by any conventional method. Polyclonal antibodiesinclude serum containing the polyclonal antibodies, as well as thefraction containing the polyclonal antibodies may be isolated from theserum. Immunoglobulin G or M can be prepared from a fraction whichrecognizes only the polypeptide of the present invention using, forexample, an affinity column coupled with the polypeptide of the presentinvention, and further purifying this fraction using protein A orprotein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammalians,and more preferably myeloma cells having an acquired property for theselection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre andMilstein, Methods Enzymol 73: 3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all the other cells,with the exception of the desired hybridoma (non-fused cells), to die.Then, the standard limiting dilution is performed to screen and clone ahybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal isimmunized with an antigen for preparing hybridoma, human lymphocytessuch as those infected by EB virus may be immunized with a polypeptide,polypeptide expressing cells or their lysates in vitro. Then, theimmunized lymphocytes are fused with human-derived myeloma cells thatare capable of indefinitely dividing, such as U266, to yield a hybridomaproducing a desired human antibody that is able to bind to thepolypeptide can be obtained (Unexamined Published Japanese PatentApplication No. (JP-A) Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are extracted. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography or an affinity column to which the polypeptide of thepresent invention is coupled. The antibody of the present invention canbe used not only for purification and detection of the polypeptide ofthe present invention, but also as a candidate for agonists andantagonists of the polypeptide of the present invention. In addition,this antibody can be applied to the antibody treatment for diseasesrelated to the polypeptide of the present invention. When the obtainedantibody is to be administered to the human body (antibody treatment), ahuman antibody or-a humanized antibody is preferable for reducingimmunogenicity.

For example, transgenic animals having a repertory of human antibodygenes may be immunized with an antigen selected from a polypeptide,polypeptide expressing cells or their lysates. Antibody producing cellsare then collected from the animals and fused with myeloma cells toobtain hybridoma, from which human antibodies against the polypeptidecan be prepared (see WO92-03918, WO93-2227, WO94-02602, WO94-25585,WO96-33735 and WO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck andLarrick, Therapeutic Monoclonal Antibodies, published in the UnitedKingdom by MacMillan Publishers LTD (1990)). For example, a DNA encodingan antibody may be cloned from an immune cell, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, so long as it binds to one or more ofthe polypeptides of the invention. For instance, the antibody fragmentmay be Fab, F(ab′)₂, Fv or single chain Fv (scFv), in which Fv fragmentsfrom H and L chains are ligated by an appropriate linker (Huston et al.,Proc Natl Acad Sci USA85: 5879-83 (1988)). More specifically, anantibody fragment may be generated by treating an antibody with anenzyme, such as papain or pepsin. Alternatively, a gene encoding theantibody fragment may be constructed, inserted into an expression vectorand expressed in an appropriate host cell (see, for example, Co et al.,J Immunol 152: 2968-76 (1994); Better and Horwitz, Methods Enzymol 178:476-96 (1989); Pluckthun and Skerra, Methods Enzymol 178: 497-515(1989); Lamoyi, Methods Enzymol 121: 652-63 (1986); Rousseaux et al.,Methods Enzymol 121: 663-9 (1986); Bird and Walker, Trends Biotechnol 9:132-7 (1991)).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides forsuch modified antibodies. The modified antibody can be obtained bychemically modifying an antibody. These modification methods areconventional in the field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, between a variable region derived from nonhumanantibody and the constant region derived from human antibody, or as ahumanized antibody, comprising the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) derivedfrom human antibody and the constant region. Such antibodies can beprepared according to known technology.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by the appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis and isoelectric focusing (Antibodies: A LaboratoryManual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)),but are not limited thereto. A protein A column and protein G column canbe used as the affinity column. Exemplary protein A columns to be usedinclude, for example, Hyper D, POROS and Sepharose F. F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). The chromatographic procedures can becarried out by liquid-phase chromatography, such as HPLC and FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA) and/orimmunofluorescence maybe used to measure the antigen binding activity ofthe antibody of the invention. In ELISA, the antibody of the presentinvention is immobilized on a plate, a polypeptide of the invention isapplied to the plate, and then a sample containing a desired antibody,such as culture supernatant of antibody producing cells or purifiedantibodies, is applied. Then, a secondary antibody that recognizes theprimary antibody and is labeled with an enzyme, such as alkalinephosphatase, is applied, and the plate is incubated. Next, afterwashing, an enzyme substrate, such as p-nitrophenyl phosphate, is addedto the plate, and the absorbance is measured to evaluate the antigenbinding activity of the sample. A fragment of the polypeptide, such as aC-terminal or N-terminal fragment, may be used as the antigen toevaluate the binding activity of the antibody. BIAcore™ (Pharmacia) maybe used to evaluate the activity of the antibody according to thepresent invention.

The above methods allow for the detection or measurement of thepolypeptide of the invention, by exposing the antibody of the inventionto a sample assumed to contain the polypeptide of the invention, anddetecting or measuring the immune complex formed by the antibody and thepolypeptide.

Because the method of detection or measurement of the polypeptideaccording to the invention can specifically detect or measure apolypeptide, the method may be useful in a variety of experiments inwhich the polypeptide is used.

Antisense Polynucleotides, Small Interfering RNAs and Ribozymes

The present invention includes an antisense oligonucleotide thathybridizes with any site within the nucleotide sequence of SEQ ID NO: 1,3 or 5. This antisense oligonucleotide is preferably against at least 15continuous nucleotides of the nucleotide sequence of SEQ ID NO: 1, 3 or5. The above-mentioned antisense oligonucleotide, which contains aninitiation codon in the above-mentioned at least 15 continuousnucleotides, is even more preferred.

Derivatives or modified products of antisense oligonucleotides can alsobe used as antisense oligonucleotides. Examples of such modifiedproducts include lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioatemodifications and phosphoroamidate modifications.

The term “antisense oligonucleotides” as used herein means, not onlythose in which the nucleotides corresponding to those constituting aspecified region of a DNA or mRNA are entirely complementary, but alsothose having a mismatch of one or more nucleotides, as long as the DNAor mRNA and the antisense oligonucleotide can specifically hybridizewith the nucleotide sequence of SEQ ID NO: 1, 3 or 5.

Such polynucleotides are contained as those having, in the “at least 15continuous nucleotide sequence region”, a homology of at least 70% orhigher, preferably at 80% or higher, more preferably 90% or higher, evenmore preferably 95% or higher. The algorithm stated herein can be usedto determine the homology. Algorithms known in the art can be used todetermine the homology. Furthermore, derivatives or modified products ofthe antisense-oligonucleotides can also be used asantisense-oligonucleotides in the present invention. Examples of suchmodified products include lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioatemodifications and phosphoroamidate modifications.

Such antisense polynucleotides are useful as probes for the isolation ordetection of DNA encoding the polypeptide of the invention or as aprimer used for amplifications.

The antisense oligonucleotide derivatives of the present invention actupon cells producing the polypeptide of the invention by binding to theDNA or mRNA encoding the polypeptide, inhibiting its transcription ortranslation, promoting the degradation of the mRNA and inhibiting theexpression of the polypeptide of the invention, thereby resulting in theinhibition of the polypeptide's function.

The present invention also includes small interfering RNAs (siRNA)comprising a combination of a sense strand nucleic acid and an antisensestrand nucleic acid of the nucleotide sequence of SEQ ID NO: 1, 3 or 5.More specifically, such siRNA for suppressing the expression ofMICAL2-PV include those that target the nucleotide sequence of SEQ IDNO: 27. Alternatively, siRNA for suppressing the expression of PCOTHinclude those that target the nucleotide sequence of SEQ ID NO: 23.

The term “siRNA” refers to a double stranded RNA molecule which preventstranslation of a target mRNA. Standard techniques are used forintroducing siRNA into cells, including those wherein DNA is used as thetemplate to transcribe RNA. The siRNA comprises a sense nucleic acidsequence and an anti-sense nucleic acid sequence of the polynucleotideencoding human MICAL2-PV or PCOTH protein (SEQ ID NO: 1, 3 or 5). ThesiRNA is constructed such that a single transcript (double stranded RNA)has both the sense and complementary antisense sequences from the targetgene, e.g., a hairpin.

The nucleotide sequence of siRNAs may be designed using an siRNA design20 computer program available from the Ambion website on the world wideweb. Nucleotide sequences for tile siRNA are selected by the computerprogram based on the following protocol:

Selection of siRNA Target Sites:

1. Beginning with the AUG start codon of the object transcript, scandownstream for AA dinucleotide sequences. Record the occurrence of eachAA and the 3′ adjacent 19 nucleotides as potential siRNA target sites.Tuschl, et al. recommend against designing siRNA to the 5′ and 3′untranslated regions (UTRs) and regions near the start codon (within 75bases) as these may be richer in regulatory protein binding sites.UTR-binding proteins and/or translation initiation complexes mayinterfere with the binding of the siRNA endonuclease complex.

2. Compare the potential target sites to the human genome database andeliminate from consideration any target sequences with significanthomology to other coding sequences. The homology search can be performedusing BLAST, which can be found on the NCBI server on the world wideweb.

3. Select qualifying target sequences for synthesis. At Ambion,preferably several target sequences can be selected along the length ofthe gene for evaluation.

The antisense oligonucleotide or siRNA of the invention inhibit theexpression of the polypeptide of the invention and is thereby useful forsuppressing the biological activity of the polypeptide of the invention.The length of the antisense oligonucleotides and siRNAs is at least 10nucleotides and may be as long as the naturally occurring thetranscript. Preferably, the antisense oligonucleotides and siRNAs have19-25 nucleotides. Most preferably, the antisense oligonucleotides andsiRNAs are less than 75, 50, 25 nucleotides in length.

Also, expression-inhibitors, comprising the antisense oligonucleotide orsiRNA of the invention, are useful in the point that they can inhibitthe biological activity of the polypeptide of the invention. Therefore,a composition comprising the antisense oligonucleotide or siRNA of thepresent invention is useful in treating a cell proliferative diseasesuch as prostate cancer.

Furthermore, the present invention provides ribozymes that inhibit theexpression of the MICAL2-PV or PCOTH polypeptide of the presentinvention.

Generally, ribozymes are classified into large ribozymes and smallribozymes. A large ribozyme is known as an enzyme that cleaves thephosphate ester bond of nucleic acids. After the reaction with the largeribozyme, the reacted site consists of a 5′-phosphate and 3′-hydroxylgroup. The large ribozyme is further classified into (1) group I intronRNA catalyzing transesterification at the 5′-splice site by guanosine;(2) group II intron RNA catalyzing self-splicing through a two stepreaction via lariat structure; and (3) RNA component of the ribonucleaseP that cleaves the tRNA precursor at the 5′ site through hydrolysis. Onthe other hand, small ribozymes have a smaller size (about 40 bp)compared to the large ribozymes and cleave RNAs to generate a5′-hydroxyl group and a 2′-3′ cyclic phosphate. Hammerhead typeribozymes (Koizumi et al., FEBS LErr 228: 225 (1988)) and hairpin typeribozymes (Buzayan, Nature 323: 349 (1986); Kikuchi and Sasaki, NucleicAcids Res 19: 6751 (1992)) are included in the small ribozymes. Methodsfor designing and constructing ribozymes are known in the art (seeKoizumi et al., FEBS Lett 228: 225 (1988); Koizumi et al., Nucleic AcidsRes 17: 7059 (1989);Kikuchi and Sasaki, Nucleic Acids Res 19:6751(1992)). Thus, ribozymes inhibiting the expression of thepolypeptides of the present invention can also be constructed based ontheir sequence information (SEQ ID NO: 1, 3 or 5) and these conventionalmethods.

Ribozymes against MICAL2-PV or PCOTH gene inhibit the expression ofover-expressed MICAL2-PV or PCOTH protein and is thus useful forsuppressing the biological activity of the protein. Therefore, theribozymes are useful in treating or preventing prostate cancer.

Diagnosing Prostate Cancer

Moreover, the present invention provides a method for diagnosing cellproliferative disease such as prostate cancer using the expression levelof the polypeptides of the present invention as a diagnostic marker.

This diagnosing method comprises the steps of: (a) detecting theexpression level of the MICAL2-PV or PCOTH gene of the presentinvention; and (b) relating an elevation of the expression level toprostate cancer.

The expression levels of the MICAL2-PV or PCOTH gene in a biologicalsample can be estimated by quantifying mRNA corresponding to or proteinencoded by the MICAL2-PV or PCOTH gene. Quantification methods for mRNAare known to those skilled in the art. For example, the levels of mRNAscorresponding to the MICAL2-PV or PCOTH gene can be estimated byNorthern blotting or RT-PCR. Since the full-length nucleotide sequencesof the MICAL2-PV or PCOTH genes are shown in SEQ ID NO: 1, 3 or 5,anyone skilled in the art can design the nucleotide sequences for probesor primers to quantify the MICAL2-PV or PCOTH gene.

Also the expression level of the MICAL2-PV or PCOTH gene can be analyzedbased on the activity or quantity of protein encoded by the gene. Amethod for determining the quantity of the MICAL2-PV or PCOTH protein isshown in bellow. For example, immunoassay method is useful for thedetermination of the proteins in biological materials. Any biologicalmaterials can be used as the biological sample for the determination ofthe protein or it's activity so long as the marker gene (MICAL2-PV orPCOTH gene) is expressed in the sample of a prostate cancer patient. Forexample, prostate duct epithelium can be mentioned as such biologicalsample. However, bodily fluids such as blood and urine may be alsoanalyzed. On the other hand, a suitable method can be selected for thedetermination of the activity of a protein encoded by the MICAL2-PV orPCOTH gene according to the activity of each protein to be analyzed.

Expression levels of the MICAL2-PV or PCOTH gene in a biological sampleare estimated and compared with those in a normal sample (sample derivedfrom a non-diseased subject). When such a comparison shows that theexpression level of the target gene is higher than those in the normalsample, the subject is judged to be affected with prostate cancer. Theexpression level of MICAL2-PV or PCOTH gene in the biological samplesfrom a normal subject and subject to be diagnosed may be determined atthe same time. Alternatively, normal ranges of the expression levels canbe determined by a statistical method based on the results obtained byanalyzing the expression level of the gene in samples previouslycollected from a control group. A result obtained by comparing thesample of a subject is compared with the normal range; when the resultdoes not fall within the normal range, the subject is judged to beaffected with or is at risk of developing prostate cancer.

In the present invention, a diagnostic agent for diagnosing cellproliferative disease, such as prostate cancer, is also provided. Thediagnostic agent of the present invention comprises a compound thatbinds to a polynucleotide or a polypeptide of the present invention.Preferably, an oligonucleotide that hybridizes to the polynucleotide ofthe present invention or an antibody that binds to the polypeptide ofthe present invention may be used as such a compound.

The present method of diagnosing prostate cancer may be applied forassessing the efficacy of treatment of prostate cancer in a subject.According to the method, a biological sample, such as a test cellpopulation, is obtained from a subject undergoing treatment for prostatecancer. The method for assessment can be conducted according toconventional methods of diagnosing prostate cancer.

If desired, biological samples are obtained from the subject at varioustime points before, during or after the treatment. The expression levelof MICAL2-PV or PCOTH gene, in the biological sample is then determinedand compared to a control level derived, for example, from a referencecell population which includes cells whose state of prostate cancer(i.e., cancerous cell or non-cancerous cell) is known. The control levelis determined in a biological sample that has not been exposed to thetreatment.

If the control level is derived from a biological sample which containsno cancerous cell, a similarity between the expression level in thesubject-derived biological sample and the control level indicates thatthe treatment is efficacious. A difference between the expression levelof the MICAL2-PV or PCOTH gene in the subject-derived biological sampleand the control level indicates a less favorable clinical outcome orprognosis.

The term “efficacious” refers that the treatment leads to a reduction inthe expression of a pathologically up-regulated gene (MICAL2-PV or PCOTHgene) or a decrease in size, prevalence or proliferating potential ofprostate cancer cells in a subject. When a treatment is appliedprophylactically, “efficacious” indicates that the treatment retards orprevents occurrence of prostate cancer. The assessment of prostatecancer can be made using standard clinical protocols. Furthermore, theefficaciousness of a treatment is determined in association with anyknown method for diagnosing or treating prostate cancer.

Moreover, the present method of diagnosing prostate cancer may also beapplied for assessing the prognosis of a subject with prostate cancer bycomparing the expression level of MICAL2-PV or PCOTH gene in apatient-derived biological sample, such as test cell population, to acontrol level. Alternatively, the expression level of MICAL2-PV or PCOTHgene in a biological sample derived from patients may be measured over aspectrum of disease stages to assess the prognosis of the patient.

An increase in the expression level of MICAL2-PV or PCOTH gene comparedto a normal control level indicates less favorable prognosis. A decreasein the expression level of MICAL2-PV or PCOTH gene indicates a morefavorable prognosis for the patient.

Screening Compounds

Using the MICAL2-PV or PCOTH gene, proteins encoded by the gene ortranscriptional regulatory region of the gene, compounds can be screenedthat alter the expression of the gene or the biological activity of apolypeptide encoded by the gene. Such compounds are expected to serve aspharmaceuticals for treating or preventing prostate cancer.

Therefore, the present invention provides a method of screening for acompound for treating or preventing prostate cancer using thepolypeptide of the present invention. An embodiment of this screeningmethod comprises the steps of: (a) contacting a test compound with apolypeptide of the present invention; (b) detecting the binding activitybetween the polypeptide of the present invention and the test compound;and (c) selecting the compound that binds to the polypeptide of thepresent invention.

The polypeptide of the present invention to be used for screening may bea recombinant polypeptide or a protein derived from the nature or apartial peptide thereof. The polypeptide of the present invention to becontacted with a test compound can be, for example, a purifiedpolypeptide, a soluble protein, a form bound to a carrier or a fusionprotein fused with other polypeptides.

As a method of screening for proteins, for example, that bind to thepolypeptide of the present invention using the polypeptide of thepresent invention, many methods well known by a person skilled in theart can be used. Such a screening can be conducted by, for example,immunoprecipitation method, specifically, in the following manner. Thegene encoding the polypeptide of the present invention is expressed inanimal cells and so on by inserting the gene to an expression vector forforeign genes, such as pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8. Thepromoter to be used for the expression may be any promoter that can beused commonly and include, for example, the SV40 early promoter (Rigbyin Williamson (ed.), Genetic Engineering, vol. 3. Academic Press,London, 83-141 (1982)), the EF-α promoter (Kim et al., Gene 91: 217-23(1990)), the CAG promoter (Niwa et al., Gene 108: 193-200 (1991)), theRSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)) theSRα promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), the CMVimmediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84:3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol ApplGenet 1: 385-94 (1982)), the Adenovirus late promoter (Kaufman et al.,Mol Cell Biol 9: 946 (1989)), the HSV TK promoter and so on. Theintroduction of the gene into animal cells to express a foreign gene canbe performed according to any methods, for example, the electroporationmethod (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calciumphosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)),the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17(1984); Sussman and Milman, Mol Cell Biol 4: 1642-3 (1985)), theLipofectin method (Derijard, B Cell 7: 1025-37 (1994); Lamb et al.,Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4(1993)) and so on. The polypeptide of the present invention can beexpressed as a fusion protein comprising a recognition site (epitope) ofa monoclonal antibody by introducing the epitope of the monoclonalantibody, whose specificity has been revealed, to the N- or C-terminusof the polypeptide of the present invention. A commercially availableepitope-antibody system can be used (Experimental Medicine 13: 85-90(1995)). Vectors which can express a fusion protein with, for example,β-galactosidase, maltose binding protein, glutathione S-transferase,green florescence protein (GFP) and so on by the use of its multiplecloning sites are commercially available.

A fusion protein prepared by introducing only small epitopes consistingof several to a dozen amino acids so as not to change the property ofthe polypeptide of the present invention by the fusion is also reported.Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, humanc-myc, FLAG Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag),E-tag (an epitope on monoclonal phage) and such, and monoclonalantibodies recognizing them can be used as the epitope-antibody systemfor screening proteins binding to the polypeptide of the presentinvention (Experimental Medicine 13: 85-90 (1995)).

In immunoprecipitation, an immune complex is formed by adding theseantibodies to cell lysate prepared using an appropriate detergent. Theimmune complex consists of the polypeptide of the present invention, apolypeptide comprising the binding ability with the polypeptide, and anantibody. Immunoprecipitation can be also conducted using antibodiesagainst the polypeptide of the present invention, besides usingantibodies against the above epitopes, which antibodies can be preparedas described above.

An immune complex can be precipitated, for example by Protein Asepharose or Protein G sepharose when the antibody is a mouse IgGantibody. If the polypeptide of the present invention is prepared as afusion protein with an epitope, such as GST, an immune complex can beformed in the same manner as in the use of the antibody against thepolypeptide of the present invention, using a substance specificallybinding to these epitopes, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, forexample, the methods in the literature (Harlow and Lane, Antibodies,511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteinsand the bound protein can be analyzed by the molecular weight of theprotein using gels with an appropriate concentration. Since the proteinbound to the polypeptide of the present invention is difficult to detectby a common staining method, such as Coomassie staining or silverstaining, the detection sensitivity for the protein can be improved byculturing cells in culture medium containing radioactive isotope,³⁵S-methionine or ³⁵S-cystein, labeling proteins in the cells, anddetecting the proteins. The target protein can be purified directly fromthe SDS-polyacrylamide gel and its sequence can be determined, when themolecular weight of a protein has been revealed.

As a method for screening proteins binding to the polypeptide of thepresent invention using the polypeptide, for example, West-Westernblotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used.Specifically, a protein binding to the polypeptide of the presentinvention can be obtained by preparing a cDNA library from cells,tissues, organs (for example, tissues such as testis for MICAL2-PV, andtestis or prostate for PCOTH), or cultured cells (e.g., LNCaP, PC3,DU145) expected to express a protein binding to the polypeptide of thepresent invention using a phage vector (e.g., ZAP), expressing theprotein on LB-agarose, fixing the protein expressed on a filter,reacting the purified and labeled polypeptide of the present inventionwith the above filter, and detecting the plaques expressing proteinsbound to the polypeptide of the present invention according to thelabel. The polypeptide of the invention may be labeled by utilizing thebinding between biotin and avidin, or by utilizing an antibody thatspecifically binds to the polypeptide of the present invention, or apeptide or polypeptide (for example, GST) that is fused to thepolypeptide of the present invention. Methods using radioisotope orfluorescence arid such may be also used.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing ceils may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, i “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP™ Two-HybridVector System” (Stratagene); the references “Dalton and Treisman, Cell68:597-612 (1992)”, “Fields and Stemglanz, Trends Genet 10:286-92(1994)”).

In the two-hybrid system, the polypeptide of the invention is fused tothe SRF-binding region or GAL4-binding region and expressed in yeastcells. A cDNA library is prepared from cells expected to express aprotein binding to the polypeptide of the invention, such that thelibrary, when expressed, is fused to the VP16 or GAL4 transcriptionalactivation region. The cDNA library is then introduced into the aboveyeast cells and the cDNA derived from the library is isolated from thepositive clones detected (when a protein binding to the polypeptide ofthe invention is expressed in yeast cells, the binding of the twoactivates a reporter gene, making positive clones detectable). A proteinencoded by the cDNA can be prepared by introducing the cDNA isolatedabove to E. coli and expressing the protein.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene and such can be used in addition to the HIS3 gene.

A compound binding to the polypeptide of the present invention can alsobe screened using affinity chromatography. For example, the polypeptideof the invention may be immobilized on a carrier of an affinity column,and a test compound, containing a protein capable of binding to thepolypeptide of the invention, is applied to the column. A test compoundherein may be, for example, cell extracts, cell lysates, etc. Afterloading the test compound, the column is washed, and compounds bound tothe polypeptide of the invention can be prepared.

When the test compound is a protein, the amino acid sequence of theobtained protein is analyzed, an oligo DNA is synthesized based on thesequence, and cDNA libraries are screened using the oligo DNA as a probeto obtain a DNA encoding the protein.

A biosensor using the surface plasmon resonance phenomenon may be usedas a mean for detecting or quantifying the bound compound in the presentinvention. When such a biosensor is used, the interaction between thepolypeptide of the invention and a test compound can be observedreal-time as a surface plasmon resonance signal, using only a minuteamount of polypeptide and without labeling (for example, BIAcore™,Pharmacia). Therefore, it is possible to evaluate the binding betweenthe polypeptide of the invention and a test compound using a biosensorsuch as BIAcore™.

The methods of screening for molecules that bind when the immobilizedpolypeptide of the present invention is exposed to synthetic chemicalcompounds, or natural substance banks or a random phage peptide displaylibrary, and the methods of screening using high-throughput based oncombinatorial chemistry techniques (Wrighton et al., Science 273: 458-64(1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9(1996)) to isolate not only proteins but chemical compounds that bind tothe protein of the present invention (including agonist and antagonist)are well known to one skilled in the art.

Alternatively, the present invention provides a method of screening fora compound for treating or preventing prostate cancer using thepolypeptide of the present invention comprising the steps as follows:

(a) contacting a test compound with the polypeptide of the presentinvention;

(b) detecting the biological activity of the polypeptide of step (a);and

(c) selecting a compound that suppresses the biological activity of thepolypeptide in comparison with the biological activity detected in theabsence of the test compound.

Since the MICAL2-PV and PCOTH proteins of the present invention have theactivity of promoting cell proliferation of prostate cancer cells, acompound which promotes or inhibits this activity of one of theseproteins of the present invention can be screened using this activity asan index.

Any polypeptides can be used for screening so long as they comprise thebiological activity of the MICAL2-PV or PCOTH protein. Such biologicalactivity include cell-proliferating activity of the human MICAL2-PV orPCOTH protein, the activity of MICAL2-PV to bind to actin. For example,a human MICAL2-PV or PCOTH protein can be used and polypeptidesfunctionally equivalent to these proteins can also be used. Suchpolypeptides may be expressed endogenously or exogenously by cells.

The compound isolated by this screening is a candidate for agonists orantagonists of the polypeptide of the present invention. The term“agonist” refers to molecules that activate the function of thepolypeptide of the present invention by binding thereto. Likewise, theterm “antagonist” refers to molecules that inhibit the function of thepolypeptide of the present invention by binding thereto. Moreover, acompound isolated by this screening is a candidate for compounds whichinhibit the in vivo interaction of the polypeptide of the presentinvention with molecules (including DNAs and proteins).

When the biological activity to be detected in the present method iscell proliferation, it can be detected, for example, by preparing cellswhich express the polypeptide of the present invention, culturing thecells in the presence of a test compound, and determining the speed ofcell proliferation, measuring the cell cycle and such, as well as bymeasuring the colony forming activity as described in the Examples.

In a further embodiment, the present invention provides methods forscreening compounds for treating or preventing prostate cancer. Asdiscussed in detail above, by controlling the expression levels of theMICAL2-PV or PCOTH, one can control the onset and progression ofprostate cancer. Thus, compounds that may be used in the treatment orprevention of prostate cancer can be identified through screenings thatuse the expression levels of MICAL2-PV or PCOTH as indices. In thecontext of the present invention, such screening may comprise, forexample, the following steps:

-   -   a) contacting a test compound with a cell expressing the        MICAL2-PV or PCOTH, and    -   b) selecting a compound that reduces the expression level of        MICAL2-PV or PCOTH in comparison with the expression level        detected in the absence of the test compound.

Cells expressing at least one of the MICAL2-PV or PCOTH include, forexample, cell lines established from prostate cancers; such cells can beused for the above screening of the present invention (e.g., LNCaP, PC3,DU145). The expression level can be estimated by methods well known toone skilled in the art. In the method of screening, a compound thatreduces the expression level of at least one of MICAL2-PV or PCOTH canbe selected as candidate agents to be used for the treatment orprevention of prostate cancer.

Alternatively, the screening method of the present invention maycomprise the following steps:

-   -   a) contacting a test compound with a cell into which a vector        comprising the transcriptional regulatory region of one or more        marker genes and a reporter gene that is expressed under the        control of the transcriptional regulatory region has been        introduced, wherein the one or more marker genes are MICAL2-PV        and PCOTH,    -   b) measuring the activity of said reporter gene; and    -   c) selecting a compound that reduces the expression level of        said reporter gene as compared to a control.

Suitable reporter genes and host cells are well known in the art. Thereporter construct required for the screening can be prepared by usingthe transcriptional regulatory region of a marker gene. When thetranscriptional regulatory region of a marker gene has been known tothose skilled in the art, a reporter construct can be prepared by usingthe previous sequence information. When the transcriptional regulatoryregion of a marker gene remains unidentified, a nucleotide segmentcontaining the transcriptional regulatory region can be isolated from agenome library based on the nucleotide sequence information of themarker gene.

In a further embodiment of the method for screening a compound fortreating or preventing prostate cancer of the present invention, themethod utilizes the binding ability of MICAL2-PV to actin. The MICAL2-PVprotein of the present invention was revealed to comprise a domainhaving homology to calponin domain, an actin-binding domain. Therefore,the protein encoded by MICAL2-PV is predicted to interact with actin orother microtubles. These domains cross-link actin filaments into bundlesand networks. The finding suggest that the MICAL2-PV protein of thepresent invention exerts the function of cell proliferation via itsbinding to molecules, such as acting and other microtubles. Thus, it isexpected that the inhibition of the binding between the MICAL2-PVprotein and actin or other microtubles leads to the suppression of cellproliferation, and compounds inhibiting the binding serve aspharmaceuticals for treating or preventing prostate cancer.

This screening method includes the steps of: (a) contacting a MICAL-PVpolypeptide of the present invention with actin in the presence of atest compound; (b) detecting the binding between the polypeptide andactin; and (c) selecting the compound that inhibits the binding betweenthe polypeptide and actin.

The MICAL2-PV polypeptide of the present invention and actin to be usedfor the screening may be a recombinant polypeptide or a protein derivedfrom the nature, or may also be a partial peptide thereof so long as itretains the binding ability to each other. The MICAL2-PV polypeptide andactin to be used in the screening can be, for example, a purifiedpolypeptide, a soluble protein, a form bound to a carrier or a fusionprotein fused with other polypeptides.

As a method of screening for compounds that inhibit the binding betweenthe MICAL2-PV protein and actin, many methods well known by one skilledin the art can be used. Such a screening can be carried out as an invitro assay system, for example, in a cellular system. Morespecifically, first, either the MICAL2-PV polypeptide or actin is boundto a support, and the other protein is added together with a testcompound thereto. Next, the mixture is incubated, washed and the otherprotein bound to the support is detected and/or measured.

Examples of supports that may be used for binding proteins includeinsoluble polysaccharides, such as agarose, cellulose and dextran; andsynthetic resins, such as polyacrylamide, polystyrene and silicon;preferably commercial available beads and plates (e.g., multi-wellplates, biosensor chip, etc.) prepared from the above materials may beused. When using beads, they bay be filled into a column.

The binding of a protein to a support may be conducted according toroutine methods, such as chemical bonding and physical adsorption.Alternatively, a protein may be bound to a support via antibodiesspecifically recognizing the protein. Moreover, binding of a protein toa support can be also conducted by means of avidin and biotin.

The binding between proteins is carried out in buffer, for example, butare not limited to, phosphate buffer and Tris buffer, as long as thebuffer does not inhibit the binding between the proteins.

In the present invention, a biosensor using the surface plasmonresonance phenomenon may be used as a mean for detecting or quantifyingthe bound protein. When such a biosensor is used, the interactionbetween the proteins can be observed real-time as a surface plasmonresonance signal, using only a minute amount of polypeptide and withoutlabeling (for example, BIAcore™, Pharmacia). Therefore, it is possibleto evaluate the binding between the MICAL2-PV polypeptide and actinusing a biosensor such as BIAcore™.

Alternatively, either the MICAL2-PV polypeptide or actin may be labeled,and the label of the bound protein may be used to detect or measure thebound protein. Specifically, after pre-labeling one of the proteins, thelabeled protein is contacted with the other protein in the presence of atest compound, and then bound proteins are detected or measuredaccording to the label after washing.

Labeling substances such as radioisotope (erg., ³H, ¹⁴C, ³²P, ³³P, ³⁵S,¹²⁵I, ¹³¹I), enzymes (e.g., alkaline phosphatase, horseradishperoxidase, β-galactosidase, β-glucosidase), fluorescent substances(e.g., fluorescein isothiosyanete (FITC), rhodamine) and biotin/avidin,may be used for the labeling of a protein in the present method. Whenthe protein is labeled with radioisotope, the detection or measurementcan be carried out by liquid scintillation. Alternatively, proteinslabeled with enzymes can be detected or measured by adding a substrateof the enzyme to detect the enzymatic change of the substrate, such asgeneration of color, with absorptiometer. Further, in case where afluorescent substance is used as the label, the bound protein may bedetected or measured using fluorophotometer.

Furthermore, the binding of the MICAL2-PV polypeptide and actin can bealso detected or measured using antibodies to the MICAL2-PV polypeptideand actin. For example, after contacting the MICAL2-PV polypeptideimmobilized on a support with a test compound and actin, the mixture isincubated and washed, and detection or measurement can be conductedusing an antibody against actin. Alternatively, actin may be immobilizedon a support, and an antibody against MICAL2-PV may be used as theantibody.

In case of using an antibody in the present screening, the antibody ispreferably labeled with one of the labeling substances mentioned above,and detected or measured based on the labeling substance. Alternatively,the antibody against the MICAL2-PV polypeptide or actin may be used as aprimary antibody to be detected with a secondary antibody that islabeled with a labeling substance. Furthermore, the antibody bound tothe protein in the screening of the present invention may be detected ormeasured using protein G or protein A column.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing ceils may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, i “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP™ Two-HybridVector System” (Stratagene); the references “Dalton and Treisman, Cell68:597-612 (1992)”, “Fields and Stemglanz, Trends Genet 10:286-92(1994)”).

In the two-hybrid system, the MICAL2-PV polypeptide of the invention isfused to the SRF-binding region or GAL4-binding region and expressed inyeast cells. The actin binding to the MICAL2-PV polypeptide of theinvention is fused to the VP16 or GAL4 transcriptional activation regionand also expressed in the yeast cells in the existence of a testcompound. When the test compound does not inhibit the binding betweenthe MICAL2-PV polypeptide and actin, the binding of the two activates areporter gene, making positive clones detectable.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene and such can be used besides HIS3 gene.

Any test compound, for example, cell extracts, cell culture supernatant,products of fermenting microorganism, extracts from marine organism,plant extracts, purified or crude proteins, peptides, non-peptidecompounds, synthetic micromolecular compounds and natural compounds canbe used in the screening methods of the present invention. The testcompound of the present invention can be also obtained using any of thenumerous approaches in combinatorial library methods known in the art,including (1) biological libraries, (2) spatially addressable parallelsolid phase or solution phase libraries, (3) synthetic library methodsrequiring deconvolution, (4) the “one-bead one-compound” library methodand (5) synthetic library methods using affinity chromatographyselection. The biological library methods using affinity chromatographyselection is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries canbe found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermannet al. (1994) J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059; Carellet al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; Gallop et al. (1994)J. Med. Chem. 37: 1233). Libraries of compounds may be presented insolution (see Houghten (1992) Bio/Techniques 13: 412) or on beads (Lam(1991) Nature 354: 82), chips (Fodor (1993) Nature 364: 555), bacteria(U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484,and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89: 1865) or phage (Scott and Smith (1990) Science 249: 386; Delvin(1990) Science 249: 404; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87: 6378; Felici (1991) J. Mol. Biol. 222: 301; US Pat. Application2002103360).

A compound isolated by the screening methods of the present invention isa candidate for drugs which promote or inhibit the activity of thepolypeptide of the present invention, for treating or preventingdiseases attributed to, for example, cell proliferative diseases, suchas prostate cancer. A compound in which a part of the structure of thecompound obtained by the present screening methods of the presentinvention is converted by addition, deletion and/or replacement, isincluded in the compounds obtained by the screening methods of thepresent invention.

Pharmaceutical Compositions for Treating or Preventing Prostate Cancer

The present invention provides compositions for treating or preventingprostate cancer comprising any of the compounds selected by thescreening methods of the present invention.

When administrating a compound isolated by the screening methods of thepresent invention as a pharmaceutical for humans or other mammals, suchas mice, rats, guinea-pigs, rabbits, cats, dogs, sheep, pigs, cattle,monkeys, baboons, chimpanzees, for treating a cell proliferative disease(e.g., prostate cancer) the isolated compound can be directlyadministered or can be formulated into a dosage form using knownpharmaceutical preparation methods. For example, according to the need,the drugs can be taken orally, as sugarcoated tablets, capsules, elixirsand microcapsules; or non-orally, in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be mixed withpharmacologically acceptable carriers or medium, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,suspending agents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives, binders and such, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples of additives that can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier, such as oil, can also befurther included in the above ingredients. Sterile composites forinjections can be formulated following normal drug implementations usingvehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannnose, D-mannitol and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM)and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizers and may be formulated with a buffer, such as phosphatebuffer and sodium acetate buffer; a pain-killer, such as procainehydrochloride; a stabilizer, such as benzyl alcohol, phenol; and ananti-oxidant. The prepared injection may be filled into a suitableampule.

Methods well known to one skilled in the art may be used to administerthe inventive pharmaceutical compound to patients, for example asintraarterial, intravenous, percutaneous injections and also asintranasal, transbronchial, intramuscular or oral administrations. Thedosage and method of administration vary according to the body-weightand age of a patient and the administration method; however, one skilledin the art can routinely select them. If said compound is encodable by aDNA, the DNA can, be inserted into a vector for gene therapy and thevector administered to perform the therapy. The dosage and method ofadministration vary according to the body-weight, age, and symptoms of apatient but one skilled in the art can select them suitably.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with the polypeptide of thepresent invention and regulates its activity is about 0.1 mg to about100 mg per day, preferably about 1.0 mg to about 50 mg per day and morepreferably about 1.0 mg to about 20 mg per day, when administered orallyto a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms and method of administration, it isconvenient to intravenously inject a dose of about 0.01 mg to about 30mg per day, preferably about 0.1 to about 20 mg per day and morepreferably about 0.1 to about 10 mg per day. Also, in the case of otheranimals too, it is possible to administer an amount converted to 60 kgsof body-weight.

Furthermore, the present invention provides pharmaceutical compositionsfor treating or preventing prostate cancer comprising active ingredientsthat inhibits the expression of MICAL2-PV or PCOTH gene. Such activeingredients include antisense polynucleotides, siRNAs or ribozymesagainst the MICAL2-PV or PCOTH gene or derivatives, such as expressionvector, of the antisense polynucleotides, siRNAs or ribozymes.

These active ingredients can be made into an external preparation, suchas a liniment or a poultice, by mixing with a suitable base materialwhich is inactive against the derivatives. Also, as needed, they can beformulated into tablets, powders, granules, capsules, liposome capsules,injections, solutions, nose-drops and freeze-drying agents by addingexcipients, isotonic agents, solubilizers, stabilizers, preservatives,pain-killers and such. These can be prepared according to conventionalmethods.

The active ingredient is given to the patient by directly applying ontothe ailing site or by injecting into a blood vessel so that it willreach the site of ailment. A mounting medium can also be used toincrease durability and membrane-permeability. Examples of moutingmedium includes liposome, poly-L-lysine, lipid, cholesterol, lipofectinor derivatives of these.

The dosage of such compositions of the present invention can be adjustedsuitably according to the patient's condition and used in desiredamounts. For example, a dose range of 0.1 to 100 mg/kg, preferably 0.1to 50 mg/kg can be administered.

Another embodiment of the present invention is a composition fortreating or preventing prostate cancer comprising an antibody against apolypeptide encoded by the MICAL2-PV or PCOTH gene or fragments of theantibody that bind to the polypeptide.

Although there are some differences according to the symptoms, the doseof an antibody or fragments thereof for treating or preventing prostatecancer is about 0.1 mg to about 100 mg per day, preferably about 1.0 mgto about 50 mg per day and more preferably about 1.0 mg to about 20 mgper day, when administered orally to a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe condition of the patient, symptoms of the disease and method of a sedministration, it is convenient to intravenously inject a dose of about0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg perday and more preferably about 0.1 to about 10 mg per day. A the caofother animals too, it is possible to administer an amount converted to60 kg of body-weight.

Methods for Treating or Preventing Prostate Cancer

The invention provides a method for treating or preventing prostatecancer in a subject. Therapeutic compounds are administeredprophylactically or therapeutically to subject suffering from or at riskof (or susceptible to) developing prostate cancer. Such subjects areidentified using standard clinical methods or by detecting an aberrantexpression level or activity of MICAL2-PV or PCOTH. Prophylacticadministration occurs prior to the manifestation of overt clinicalsymptoms of disease, such that a disease or disorder is prevented or,alternatively, delayed in its progression.

The therapeutic method includes decreasing the expression or function,or both of MICAL2-PV or PCOTH gene. In these methods, the subject istreated with an effective amount of a compound, which decreases one orboth of the over-expressed genes (MICAL2-PV or PCOTH gene) in thesubject. Administration can be systemic or local. Therapeutic compoundsinclude compounds that decrease the expression level of such geneendogenously existing in the prostate cancerous cells (i.e., compoundsthat down-regulate the expression of the over-expressed gene(s)).Administration of such therapeutic compounds counter the effects ofaberrantly-over expressed gene(s) in the subjects cells and are expectedto improve the clinical condition of the subject. Such compounds can beobtained by the screening method of the present invention describedabove.

The expression of MICAL2-PV or PCOTH gene may be also inhibited in anyof several ways known in the art including administering to the subjecta nucleic acid that inhibits or antagonizes the expression of thegene(s). Antisense oligonucleotides, siRNA or ribozymes which disruptsexpression of the gene(s) can be used for inhibiting the expression ofthe genes.

As noted above, antisense-oligonucleotides corresponding to thenucleotide sequence of MICAL2-PV or PCOTH gene can be used to reduce theexpression level of the MICAL2-PV or PCOTH gene. Specifically, theantisense-oligonucleotides of the present invention may act by bindingto any of the polypeptides encoded by the MICAL2-PV or PCOTH gene, ormRNAs corresponding thereto, thereby inhibiting the transcription ortranslation of the genes, promoting the degradation of the mRNAs, and/orinhibiting the expression of proteins encoded by the genes, and finallyinhibiting the function of the MICAL2-PV or PCOTH proteins.

An antisense-oligonucleotides and derivatives thereof can be made intoan external preparation, such as a liniment or a poultice, by mixingwith a suitable base material which is inactive against the derivativeand used in the method for treating or preventing prostate cancer of thepresent invention.

The nucleic acids that inhibit one or more gene products ofover-expressed genes also include small interfering RNAs (siRNA)comprising a combination of a sense strand nucleic acid and an antisensestrand nucleic acid of the nucleotide sequence encoding the MICAL2-PV orPCOTH gene. Standard techniques of introducing siRNA into the cell canbe used in the treatment or prevention of the present invention,including those in which DNA is a template from which RNA istranscribed. The siRNA is constructed such that a single transcript hasboth the sense and complementary antisense sequences from the targetgene, e.g., a hairpin.

The method is used to suppress gene expression of a cell withup-regulated expression of the MICAL2-PV or PCOTH gene. Binding of thesiRNA to the MICAL2-PV or PCOTH gene transcript in the target cellresults in a reduction of MICAL2-PV or PCOTH protein production by thecell.

The nucleic acids that inhibit one or more gene products ofover-expressed genes also include ribozymes against the over-expressedgene(s) (MICAL2-PV or PCOTH gene).

Moreover, the present invention provides a method for treating orpreventing a cell proliferative disease, such as prostate cancer, usingan antibody against the polypeptide of the present invention. Accordingto the method, a pharmaceutically effective amount of an antibodyagainst the polypeptide of the present invention is administered. Sincethe expression of the MICAL2-PV and PCOTH protein are up-regulated inprostate cancer cells and the suppression of the expression of theseproteins leads to the decrease in cell proliferating activity, it isexpected that cell proliferative diseases can be treated or prevented bybinding the antibody and these proteins. Thus, an antibody against thepolypeptide of the present invention are administered at a dosagesufficient to reduce the activity of the protein of the presentinvention, which is in the range of 0.1 to about 250 mg/kg per day. Thedose range for adult humans is generally from about 5 mg to about 17.5g/day, preferably about 5 mg to about 10 g/day, and most preferablyabout 100 mg to about 3 g/day.

Alternatively, an antibody binding to a cell surface marker specific fortumor cells can be used as a tool for drug delivery. For example, theantibody conjugated with a cytotoxic agent is administered at a dosagesufficient to injure tumor cells.

The present invention also relates to a method of inducing anti-tumorimmunity comprising the step of administering MICAL2-PV or PCOTH proteinor an immunologically active fragment thereof, or a polynucleotideencoding the protein or fragments thereof. The MICAL2-PV or PCOTHprotein or the immunologically active fragments thereof are useful asvaccines against cell proliferative diseases such as prostate cancer. Insome cases the proteins or fragments thereof may be administered in aform bound to the T cell receptor (TCR) or presented by an antigenpresenting cell (APC), such as macrophage, dendritic cell (DC), orB-cells. Due to the strong antigen presenting ability of DC, the use ofDC is most preferable among the APCs.

In the present invention, vaccine against cell proliferative diseaserefers to a substance that has the function to induce anti-tumorimmunity upon inoculation into animals. In general, anti-tumor immunityincludes immune responses such as follows:lso, in

-   -   induction of cytotoxic lymphocytes against tumors,    -   induction of antibodies that recognize tumors, and    -   induction of anti-tumor cytokine production.

Therefore, when a certain protein induces any one of these immuneresponses upon inoculation into an animal, the protein is decided tohave anti-tumor immunity inducing effect. The induction of theanti-tumor immunity by a protein can be detected by observing in vivo orin vitro the response of the immune system in the host against theprotein.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. A foreign substance that enters the livingbody is presented to T cells and B cells by the action of antigenpresenting cells (APCs). T cells that respond to the antigen presentedby APC in antigen specific manner differentiate into cytotoxic T cells(or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen,and then proliferate (this is referred to as activation of T cells).Therefore, CTL induction by a certain peptide can be evaluated bypresenting the peptide to T cell by APC, and detecting the induction ofCTL. Furthermore, APC has the effect of activating CD4+ T cells, CD8+ Tcells, macrophages, eosinophils, and NK cells. Since CD4+ T cells andCD8+ T cells are also important in anti-tumor immunity, the anti-tumorimmunity inducing action of the peptide can be evaluated using theactivation effect of these cells as indicators.

A method for evaluating the inducing action of CTL using dendritic cells(DCs) as APC is well known in the art. DC is a representative APC havingthe strongest CTL inducing action among APCs. In this method, the testpolypeptide is initially contacted with DC, and then this DC iscontacted with T cells. Detection of T cells having cytotoxic effectsagainst the cells of interest after the contact with DC shows that thetest polypeptide has an activity of inducing the cytotoxic T cells.Activity of CTL against tumors can be detected, for example, using thelysis of ⁵¹Cr-labeled tumor cells as the indicator. Alternatively, themethod of evaluating the degree of tumor cell damage using ³H-thymidineuptake activity or LDH (lactose dehydrogenase)-release as the indicatoris also well known.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also beused as the APC. The induction of CTL is reported that the it can beenhanced by culturing PBMC in the presence of GM-CSF and IL-4.Similarly, CTL has been shown to be induced by culturing PBMC in thepresence of keyhole limpet hemocyanin (KLH) and IL-7.

The test polypeptides confirmed to possess CTL inducing activity bythese methods are polypeptides having DC activation effect andsubsequent CTL inducing activity. Therefore, polypeptides that induceCTL against tumor cells are useful as vaccines against tumors.Furthermore, APC that acquired the ability to induce CTL against tumorsby contacting with the polypeptides are useful as vaccines againsttumors. Furthermore, CTL that acquired cytotoxicity due to presentationof the polypeptide antigens by APC can be also used as vaccines againsttumors. Such therapeutic methods for tumors using anti-tumor immunitydue to APC and CTL are referred to as cellular immunotherapy.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction is known to increase by combining aplurality of polypeptides having different structures and contactingthem with DC. Therefore, when stimulating DC with protein fragments, itis advantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of anti-tumor immunity by a polypeptide canbe confirmed by observing the induction of antibody production againsttumors. For example, when antibodies against a polypeptide are inducedin a laboratory animal immunized with the polypeptide and when growth oftumor cells is suppressed by those antibodies, the polypeptide can bedetermined to have an ability to induce anti-tumor immunity.

Anti-tumor immunity is induced by administering the vaccine of thisinvention, and the induction of anti-tumor immunity enables treatmentand prevention of cell proliferating diseases, such as prostate cancers.Therapy against cancer or prevention of the onset of cancer includes anyof the steps, such as inhibition of the growth of cancerous cells,involution of cancer and suppression of occurrence of cancer. Decreasein mortality of individuals having cancer, decrease of tumor markers inthe blood, alleviation of detectable symptoms accompanying cancer andsuch are also included as the effect of therapy or prevention of cancer.Such therapeutic and preventive effects are preferably statisticallysignificant For example, in observation, at a significance level of 5%or less, wherein the therapeutic or preventive effect of a vaccineagainst cell proliferative diseases is compared to a control withoutvaccine administration. For example, Student's t-test, the Mann-WhitneyU-test or ANOVA may be used for statistical analyses.

The above-mentioned protein having immunological activity or a vectorencoding the protein may be combined with an adjuvant. An adjuvantrefers to a compound that enhances the immune response against theprotein when administered together (or successively) with the proteinhaving immunological activity. Examples of adjuvants include choleratoxin, salmonella toxin, alum and such, but are not limited thereto.Furthermore, the vaccine of this invention may be combined appropriatelywith a pharmaceutically acceptable carrier. Examples of such carriersare sterilized water, physiological saline, phosphate buffer, culturefluid and such. Furthermore, the vaccine may contain as necessary,stabilizers, suspensions, preservatives, surfactants and such. Thevaccine is administered systemically or locally. Vaccine administrationmay be performed by single administration or boosted by multipleadministrations.

When using APC or CTL as the vaccine of this invention, tumors can betreated or prevented, for example, by the ex vivo method. Morespecifically, PBMCs of a subject receiving treatment or preventiontherapy are collected, the cells are contacted with the polypeptide exvivo, and following the induction of APC or CTL, the cells may beadministered to the subject. APC can be also induced by introducing avector encoding the polypeptide into PBMCs ex vivo. APC or CTL inducedin vitro can be cloned prior to administration. By cloning and growingcells having high activity of damaging target cells, cellularimmunotherapy can be performed more effectively. Furthermore, APC andCTL isolated in this manner may be used for cellular immunotherapy notonly against individuals from whom the cells are derived, but alsoagainst similar types of tumors from other individuals.

Furthermore, a pharmaceutical composition for treating or preventing acell proliferative disease, such as prostate cancer, comprising apharmaceutically effective amount of the MICAL2-PV or PCOTH polypeptideis provided. The pharmaceutical composition may be used for raising antitumor immunity. The normal expression of MICAL2-PV is restricted totestis and that of PCOTH is restricted to testis and prostate.Therefore, suppression of these genes may not adversely affect otherorgans. Thus, the MICAL2-PV and PCOTH polypeptides are preferable fortreating cell proliferative disease, especially prostate cancers.Furthermore, since peptide fragments of proteins specifically expressedin cancerous cells were revealed to induce immune response against thecancer, peptide fragments of MICAL2-PV or PCOTH can also be used in apharmaceutical composition for treating or preventing cell proliferativediseases such as prostate cancers. In the present invention, thepolypeptide or fragment thereof is administered at a dosage sufficientto induce anti-tumor immunity, which is in the range of 0.1 mg to 10 mg,preferably 0.3 mg to 5 mg, more preferably 0.8 mg to 1.5 mg. Theadministrations are repeated. For example, 1 mg of the peptide orfragment thereof may be administered 4 times in every two weeks forinducing the anti-tumor immunity.

In addition, polynucleotides encoding MICAL2-PV or PCOTH, or fragmentsthereof may be used for raising anti tumor immunity. Suchpolynucleotides may be incorporated in an expression vector to expressMICAL2-PV or PCOTH, or fragments thereof in a subject to be treated.Thus, the present invention encompasses method for inducing anti tumorimmunity wherein the polynucleotides encoding MICAL2-PV or PCOTH, orfragments thereof are administered to a subject suffering or being atrisk of developing cell proliferative diseases such as prostate cancer.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Any patents, patent applications andpublications cited herein are incorporated by reference.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is illustrated in details by following Examples,but is not restricted to these Examples.

1. Materials and Methods

(1) Cell Lines and Clinical Materials

Human prostate cancer cells LNCaP, PC3 and DU145 were purchased from theAmerican Type Culture Collection (ATCC, Rockville, Md.). All cells werecultured in RPMI-1640 (Sigma, St. Louis, Mo.) for LNCap, Dulbecco'smodified Eagle's medium (Invitrogen, Carlsbad, Calif.) for DU145, andF12 nutrient mixture (Invitrogen, Carlsbad, Calif.) for PC3, eachsupplemented with 10% fetal bovine serum and 1% antibiotic/antimycoticsolution (Sigma).

(2) Isolation of Two Novel Human Genes Using cDNA Microarray

Fabrication of cDNA microarray slides has been described (Ono et al.,Cancer Res 60: 5007-11 (2000)). For each analysis of expressionprofiles, the present inventors prepared duplicate sets of cDNAmicroarray slides containing 23,040 cDNA spots, to reduce experimentalfluctuation. Briefly, total RNAs were purified from prostate cancercells and normal prostate duct epithelium microdissected from 20prostate cancer tissues. T7-based RNA amplification was carried out toobtain adequate RNA for microarray experiments. Aliquots of amplifiedRNA from prostate cancer cells and normal duct epithelium were labeledby reverse transcription with Cy5-dCTP and Cy3-dCTP, respectively(Amersham Biosciences, Buckinghamshire, UK). Hybridization, washing, anddetection were carried out as described previously (Ono et al., CancerRes 60: 5007-11 (2000)). Subsequently, among the up-regulated genes, twogenes with in-house identification number D4493 and A5736 were focuseddue to its expression ratio which was greater than 5.0 in more than 50%of informative prostate cancers and their expression level in normalvital major organs which was relatively low according to previous dataobtained by the inventors on gene expression in 29 normal human tissues(Saito-Hisaminato et al., DNA Res 9: 35-45 (2002)).

(3) Northern-blot Analysis

Human multiple-tissue Northern blots (Clontech, Palo Alto, Calif.) werehybridized with an [α-³²P] dCTP-labeled PCR product of D4493 and A5736.The PCR products were prepared by RT-PCR using primers:5′-CCGACACTCTGGGTAGGAGA-3′(SEQ.ID.NO.7) and5′-TACGTGAGCTCTGAGGACCA-3′(SEQ.ID.NO.8) for D4493; and5′-TGAAGCAACAAAGAGAGGAGGAG-3′(SEQ.ID.NO.9) and5′-CCGTGTGGCACTGTAAATGATTA-3′(SEQ.ID.NO.10) for A5736.Pre-hybridization, hybridization and washing were performed according tothe supplier's recommendations. The blots were autoradiographed withintensifying screens at −80° C. for 7 days.

(4) Semi-quantitative RT-PCR Analysis

Total RNA was extracted from cultured cells and clinical samples usingTRIzol™ Reagent (Invitrogen) according to the manufacturer's protocol.Extracted RNA was treated with DNase I (Roche) and reversely transcribedfor single-stranded eDNAs using oligo(dT)I6 primer with Superscript™ IIreverse transcriptase (Roche). Appropriate dilutions of eachsingle-stranded eDNA were prepared for subsequent PCR amplification bymonitoring the 13-actin (ACTB) as a quantitative control. The primersequences were

5′-CATCCACGAAACTACCTTCAACT-3′ (SEQ.ID.NO. 11) and5′-TCTCCTTAGAGAGAAGTGGGGTG-3′ (SEQ.ID.NO. 12) for ACTB;

5′-CCGACACTCTGGGTAGGAGA-3′ (SEQ.ID.NO. 13) and5′-TACGTGAGCTCTGAGGACCA-3′ (SEQ.ID.NO.14) for D4493; and

5′-GCAGGGATATCTTTGAGAAA-3′ (SEQ.ID.NO. 15) and5′-CCAGGATCTGCACAAATACA-3′ (SEQ.ID.NO. 16) for A5736. All reactionsinvolved initial denaturation at 94° C. for 2 min followed by 21 cycles(for ACTB) or 35 cycles (for D4493 and A5736) at 94° C. for 30 s, 58° C.for 30 s, and 72° C. for 1 min, on a GeneAmp™ PCR system 9700 (PEApplied Biosystems).

(5) Construction of Expression Vector

The entire coding sequence of D4493 cDNA was amplified by RT-PCR withprimers; 5′-CGTGGATCCC AGACCGTGCATCATGGGCAC ATCTGAAGAAGGAAACTTGC-3′(SEQ.ID.NO.17) (D4493-forward) and 5′-AATCTCGAGTCAGGGGCAGAAGGGGAATAA GG-3′(SEQ.ID.NO.18) (D4493-reverse). The productwas inserted into the EcoRI sites of pCAGGS neo vector after bluntingtreatment. For detection of D4493 protein expression, HA tag was fusedat NH₂ or COOH terminus of the D4493 protein. The entire coding sequenceof A5736 cDNA was also amplified by RT-PCR with primers; 5′-CCCAAGCTTATGGGGGAAAA CGAGGATGA-3′(SEQ.ID.NO.19) (A5736-forward) and 5′-TTTTCCTTTTGCGGCCGCGC GGAGCTTGAC TGGGAAGC-3′(SEQ.ID.NO.20) (D5736-reverse). Theproduct was inserted into the Hind III and Not I sites of pcDNA3.1(+)/myc-His (Invitrogen). These constructs were confirmed by DNAsequencing.

(6) Immunocytochemical Staining

COS7 cells were transfected transiently with pCAGGS neo-D4493 andpcDNA3.1 (-)-A5736-myc-His using FuGENE™ 6 (Roche) according tomanufacture's instruction, then were fixed with 4% paraformaldehyde andpermeablilized with 0.2% Triton X-100 in PBS for 3 min at roomtemperature. Next, the cells were covered with blocking solution (3%BSA/PBS containing 0.2% Triton X-100) for 30 min at room temperature,and incubated with a rat anti-HA antibody (Roche) or a rat anti-mycantibody (Sigma) in blocking solution for 60 min at room temperature.After washing with PBS, cells were stained by a FITC-conjugated anti-ratsecondary antibody (Organon teknika), and Rhodamine-conjugatedanti-mouse secondary antibody (ICN Biomedicals) for 60 min at roomtemperature. Specimen was mounted with VECTASHIELD (VECTOR Laboratories,Inc, Burlingame, Calif.) containing4′,6′-diamidine-2′-phenylindolendihydrochrolide (DAPI) and visualizedwith Spectral Confocal Scanning Systems (Leica).

(7) siRNA Expression Vector and Transfection to Prostate Cancer Cell

siRNA expression vector (psiU6BX) were used for evaluating the effect orRNAi to the target genes. The U6 promoter was cloned into the upstreamof the gene specific sequence (19nt sequence from the target transcriptseparated by a short spacer TTCAAGAGA from the reverse complement of thesame sequence) and five thymidines as termination signal, furthermoreneo cassette was integrated to provide resistance against Geneticin(Sigma). The target sequences for D4493 were5′-TAGGGCCCATGGGGCCCGG-3′(SEQ.ID.NO.21) (si1),5′-ACCAGTTGGGCCCAAAGGC-3′(SEQ.ID.NO.22) (si2),5′-AGGCCCAATGTTGCCCCTT-3′(SEQ.ID.NO.23) (si3),5′-TGTTGCCCCTTGGCCCCTC-3′(SEQ.ID.NO.24) (si4), and5′-GAAGCAGCACGACTTCTTC-3′(SEQ.ID.NO.25) (EGFP), respectively. The targetsequences for A5736 are 5′-GCTGCTGGCCTCCATATCA-3′(SEQ.ID.NO.26) (si1),5′-TGCTTACAACTACTGCTAC-3′(SEQ.ID.NO.27) (si2), and5′-CTACTGCTACATGTACGAG-3′(SEQ.ID.NO.28) (si3). The human prostate cancercell lines LNCaP, PC3 and DU145 were plated onto 10-cm dishes (5×10⁵cells/dish), and transfected with psiU6BX containing EGFP targetsequence (psiU6BX-EGFP) and psiH1BX containing target sequence(psiU6BX-si1˜4 of D4493 or si1˜3 of A5736) using Lipofectamine 2000(Invitrogen) according to manufacture's instruction. Cells were selectedby treating with 500 mg/ml Geneticin for one week and preliminary cellswere harvested for expression analysis of the target genes and analyzedby RT-PCR. The primers of RT-PCR were the same as described above. Thesecells were also stained by Giemsa solution and performed MTT assay.

2. Results

(1) Identification of PCOTH and Prostate Cancer Variants of MICAL2(MICAL2-PV) as Up-regulated Genes in Prostate Cancer Cells

Gene-expression profiles of purified cancer cells from 20 prostatecancers were analyzed using cDNA microarray representing 23,040 humangenes. As a result, 88 genes that were commonly up-regulated in prostatecancer cells were identified. Among the identified genes, one gene withan in-house code D4493 that was markedly up-regulated in more than 50%of prostate cancer was focused and validated for its over-expressedpattern in prostate cancer cells by RT-PCR (FIG. 1A). D4493 wasoverlapped by two ESTs (BC015452 and BG178505) derived from prostatecancer cDNA library and was revealed to be identical with LOC221179(XP_(—)167955). Comparison between mouse/rat genome sequences, a novelcoding region of LOC221179 was determined which codes a 100-amino acidprotein. Northern blot analysis demonstrated that LOC221179 was highlyand locally expressed in prostate and testis (FIG. 1B). This product hasone characteristic domain, collagen triple helix repeat (FIG. 1C), whichis a characteristic feature of the collagen superfamily. Thus, the genewas dubbed “PCOTH (prostate collagen triple helix)”.

Next another gene, A5736, also markedly up-regulated in more than 50% ofprostate cancer was focused and validated the over-expressed pattern inprostate cancer cells by RT-PCR (FIG. 2A). This gene overlapped withMICAL2 (Molecule Interacting with CasL 2). Northern blot analysis usingthe sequence of A5736 as a probe demonstrated that a transcript ofapproximately 7.5 kb was abundantly expressed in testis and prostatecancer cell lines. However, normal transcript of MICAL2 transcript wasconfirmed to have a size of 3.8 kb (FIG. 2B). To solve this discrepancyof size, RACE was performed to identify unknown transcribed region. As aresult of RACE using testis cDNA, novel variants of MICAL2 with a sizeof 7.5 kb were identified. The coding region of the 3′ terminus of theidentified variants was different from MICAL2 and encoded a 976amino-acid residue instead of the 1124 amino-acid residue of the normalMICAL2 protein (FIG. 2C). According to the invention, the presentinventors discovered two novel variants of MICAL2, one long form variant(Accession number: AB 110785) and one short form variant (Accessionnumber: AB110786) wherein one exon is spliced out from the long formvariant. Herein, the variants are collectively called “MICAL2-PV(MICAL2prostate cancer-variants)”.

(2) Subcellular Localization

To further investigate the subcellular localization of PCOTH andMICAL2-PV proteins, these proteins were ligated with tag and weretransiently over-expressed in COS7 cells to perform immunocytochemicalstaining. As shown in FIG. 3, exogenous PCOTH-HA protein was localizedin the cell membrane or submembrane (FIG. 3A), and exogenousMICAL2-PV-Myc protein was localized in the cytoplasma of COS7 cells(FIG. 3B).

(3) Growth Suppression Mediated by siRNA in Prostate Cancer Cell Lines

To investigate the effect of over-expression of these genes on thegrowth or survival of prostate cancer cells, endogenous expression ofthese genes were specifically knocked down by the mammalian vector-basedRNA interference (RNAi) technique. Transfection of siRNA-producingvectors of some of the designed siRNA for PCOTH (FIG. 4A) and MICAL2-PV(FIG. 4D) resulted in reduction of endogenous expression. Theknocking-down effect by the siRNA on the transcript of PCOTH resulted ina drastic growth suppression in the colony formation assay and MTT assay(FIGS. 4B and 4C). The knocking-down effect by the siRNA on thetranscript of MICAL2-PV also resulted in growth suppression in thecolony formation assay (FIG. 4E). These findings strongly suggest thatover-expression of PCOTH and MICAL2-PV in prostate cancer cells isassociated with cancer cell growth and that these genes or proteinsencoded by the genes are promising molecular targets for prostate cancertherapy wherein the genes are blocked or knocked down.

INDUSTRIAL APPLICABILITY

The expression of human genes MICAL2-PV and PCOTH is markedly elevatedin prostate cancer as compared to non-cancerous prostate ductepithelium. Accordingly, these genes may serve as a diagnostic marker ofprostate cancer and the proteins encoded thereby may be used indiagnostic assays of prostate cancer.

The present inventors have also shown that the expression of novelprotein MICAL2-PV or PCOTH promotes cell growth whereas cell growth issuppressed by small interfering RNAs corresponding to the MICAL2-PV orPCOTH gene. These findings suggest that each of MICAL2-PV and PCOTHproteins stimulate oncogenic activity. Thus, each of these noveloncoproteins is a useful target for the development of anti-cancerpharmaceuticals. For example, agents that block the expression ofMICAL2-PV or PCOTH, or prevent its activity may find therapeutic utilityas anti-cancer agents, particularly anti-cancer agents for the treatmentof prostate cancers. Examples of such agents include antisenseoligonucleotides, small interfering RNAs, and ribozymes against theMICAL2-PV or PCOTH gene, and antibodies that recognize MICAL2-PV orPCOTH.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

1. A substantially pure polypeptide comprising the entirety of the aminoacid sequence of SEQ ID NO:
 4. 2. A method for producing the polypeptideof claim 1, said method comprising the steps of: (a) culturing a hostcell comprising: (1) a polynucleotide encoding the polypeptide; or (2) avector comprising the polynucleotide encoding the polypeptide; (b)allowing the host cell to express the polypeptide; and (c) collectingthe expressed polypeptide.